eNOS mutations useful for gene therapy and therapeutic screening

ABSTRACT

The present invention relates to new NOS variants or mutants which contain structural alterations in the site of Akt dependent phosphorylation. The altered NOS proteins or peptides, especially the human eNOS proteins or peptides, Akt proteins or polypeptides and their encoding nucleic acid molecules are useful as gene therapy agents for the treatment of diseases including post angioplasty restenosis, hypertension, atherosclerosis, heart failure, diabetes and diseases with defective angiogenesis. NOS proteins and peptides are also useful in methods of screening for agents which modulate NOS activity.

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/129,550, filed Apr. 16, 1999, which is hereinincorporated by reference in its entirety.

ACKNOWLEDGMENT OF FEDERAL SUPPORT

[0002] The present invention arose in part from research funded by thefollowing federal grant monies: HL 57665 and HL 61371.

TECHNICAL FIELD

[0003] The present invention relates to new NOS variants or mutantswhich contain structural alterations in the site of Akt dependentphosphorylation. The altered NOS proteins or peptides and their encodingnucleic acid molecules are useful as gene therapy agents for thetreatment of diseases including post angioplasty restenosis,hypertension, atherosclerosis, heart failure, diabetes and diseases withdefective angiogenesis.

BACKGROUND OF THE INVENTION

[0004] Atherosclerosis and vascular thrombosis are a major cause ofmorbidity and mortality, leading to coronary artery disease, myocardialinfarction, and stroke. Atherosclerosis begins with an alteration in theendothelium which lines the blood vessels. An endothelial alteration mayeventually result in the development of an endothelial lesion caused, inpart, by the uptake of oxidized low-density lipoprotein (LDL)cholesterol. Rupture of this lesion can lead to thrombosis and occlusionof the blood vessel. In the case of a coronary artery, rupture of acomplex lesion may precipitate a myocardial infarction, whereas in thecase of a carotid artery, stroke may ensue.

[0005] In atherosclerotic coronary heart disease, endothelialdysfunction may diminish production of vasodilatory substances, such asnitric oxide. Myocardial ischemia results when autoregulatoryvasodilation is prevented, whether by flow-limiting coronary arterialstenosis or by endothelial dysfunction. In both cases, arterial bloodflow can no longer increase proportional to rising oxygen demands. Inother situations, myocardial ischemia may occur when oxygen demands areconstant but there is a primary decrease in coronary blood flow mediatedvia coronary artery spasm, rapid evolution of the underlyingatherosclerotic plaque leading to a reduced coronary arterial lumencaliber, and/or intermittent microvascular plugging by plateletaggregates.

[0006] Balloon angioplasty is commonly used to reopen a blood vesselwhich is narrowed by plaque. Although balloon angioplasty is successfulin a high percentage of the cases in opening the vessel, it oftendenudes the endothelium and injures the vessel in the process. Thisdamage causes the migration and proliferation of vascular smooth musclecells of the blood vessel into the area of injury to form a lesion,known as myointimal hyperplasia or restenosis. This new lesion leads toa recurrence of symptoms within three to six months after theangioplasty in a significant proportion of patients.

[0007] In atherosclerosis, thrombosis and restenosis there is also aloss of normal vascular function, such that vessels tend to constrict,rather than dilate. The excessive vasoconstriction of the vessel causesfurther narrowing of the vessel lumen, limiting blood flow. This cancause symptoms such as angina (if a heart artery is involved), ortransient cerebral ischemia (i.e. a “small stroke”, if a brain vessel isinvolved). This abnormal vascular function (excessive vasoconstrictionor inadequate vasodilation) occurs in other disease states as well.Hypertension (high blood pressure) is caused by excessivevasoconstriction, as well as thickening, of the vessel wall,particularly in the smaller vessels of the circulation. This process mayaffect the lung vessels as well causing pulmonary (lung) hypertension.Other disorders known to be associated with excessive vasoconstriction,or inadequate vasodilation include transplant atherosclerosis,congestive heart failure, toxemia of pregnancy, Raynaud's phenomenon,Prinzmetal's angina (coronary vasospasm), cerebral vasospasm,hemolytic-uremia and impotence.

[0008] A substance released by the endothelium, initially referred to as“endothelium derived relaxing factor” (EDRF), plays an important role ininhibiting these pathologic processes. EDRF is now known to be nitricoxide (NO). NO plays many roles in human physiology, including therelaxation of vascular smooth muscle, the inhibition of plateletaggregation, the inhibition of mitogenesis, the proliferation ofvascular smooth muscle, and leukocyte adherence. Because NO is the mostpotent endogenous vasodilator, and because it is largely responsible forexercise-induced vasodilation in the conduit arteries, enhancement of NOsynthesis could also improve exercise capacity in normal individuals andthose with vascular disease.

[0009] Endothelial nitric oxide synthase (eNOS) is the nitric oxidesynthase (NOS) isoform responsible for the maintenance of systemic bloodpressure, vascular remodeling and angiogenesis (Shesely et al., 1996;Huang et al., 1995; Rudic et al., 1998; Murohara et al., 1998). Asdeficient endothelial production of NO is an early, persistent featureof atherosclerosis and vascular injury, eNOS has proven to be anattractive target for vascular gene therapy. While the regulation ofeNOS activation remains largely undefined, it is known that eNOS isphosphorylated in response to various forms of cellular stimulation(Michel et al., 1993; Garcia-Cardena et al., 1996; Corson et al., 1996),however, the role of phosphorylation in the regulation of nitric oxide(NO) production and the kinase(s) responsible has not been previouslyelucidated.

SUMMARY OF THE INVENTION

[0010] The present inventions result, in part, from the new discoverythat the serine/threonine protein kinase, Akt (protein kinase B), candirectly phosphorylate eNOS on a serine residue corresponding to residue1179 in bovine eNOS or residue 1177 in human eNOS and activate theenzyme leading to NO production. Mutant eNOS (S1179A or S1177A) isresistant to Akt phosphorylation and activation while mutant eNOS(S1179D and S1177D) or (S1179E and S1177E) is constitutively active.Moreover, using adenoviral mediated gene transfer activated Aktincreases basal NO release from endothelial cells and activationdeficient Akt attenuates VEGF stimulated NO production. Thus, eNOS is anewly described Akt substrate linking signal transduction via Akt to therelease of the gaseous second messenger, NO. The present inventions arealso based in part on the findings that mutant eNOS (S1179D) exhibits anincrease in the rate of NO production and an increase in reductaseactivity.

[0011] The present invention includes NOS, polypeptides or proteins andtheir encoding isolated nucleic acid molecules, wherein the NOSpolypeptide or protein contains a substituted amino acid residuecorresponding to residue 1179 of bovine eNOS, residue 1177 of humaneNOS, residue 1412 of rat nNOS, or residue 1415 at human nNOS. Preferredsubstitutions include amino acids with negatively charged R groups,including aspartic acid and glutamic acid.

[0012] The present invention also includes NOS polypeptides or proteinsand their encoding isolated nucleic acid molecules, wherein the NOSpolypeptide or protein contains a substituted amino acid residuecorresponding to residue 1179 of bovine eNOS, residue 1177 of humaneNOS, residue 1412 of rat nNOS, or residue 1415 at human nNOS. Preferredsubstitutions include amino acids with non-negatively charged R groups,such as alanine.

[0013] The present invention provides methods for stimulating collateralvessel development in ischemic diseases with deficient endogenousangiogenesis, specifically peripheral vascular disease and/or myocardialischemia in a patient comprising delivering a transgene coding for anNOS polypeptide of the invention or an Akt polypeptide.

[0014] The invention further includes a non-human transgenic animalwhich express an NOS polypeptide of the invention.

[0015] Lastly, the invention includes methods of identifying an agentwhich modulates the Akt regulated activity of NOS, comprising thegeneral steps of: (a) exposing purified NOS, preferably eNOS or nNOS, ora cell that expresses NOS, preferably eNOS or nNOS, and Akt to an agent;and (b) measuring the Akt regulated activity of NOS, preferably eNOS ornNOS.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIGS. 1A-1B. Wild-type Akt, but not kinase inactive Akt increasesNO release from cells expressing membrane associated eNOS. In FIG. 1A,COS cells were transfected with plasmids for eNOS, in the absence orpresence of Akt or kinase inactive Akt (K179M) and the production of NO(assayed as NO₂ ⁻) determined by chemiluminescence. In FIG. 1B, COScells were transfected with various NOS plasmids as above. In both FIG.1A and FIG. 1B, values for NO₂ ⁻ production were subtracted from levelsobtained from cells transfected with the β-galactosidase cDNA only. Theinset shows the expression of proteins in total cell lysates. Data aremean±SEM, n=3-7 experiments; * denotes p<0.05.

[0017]FIGS. 2A-2D. Phosphorylation of eNOS by active Akt in vitro and invivo. In FIG. 2A, COS cells were transfected with HA-Akt orHA-Akt(K179M), lysates were immunoprecipitated and placed into an invitro kinase reaction with histone 2B (25 mg) or recombinant eNOS (3 mg)as substrates. The top panel depicts the incorporation of ³²P into thesubstrates and the bottom panel shows the amount of substrate byCoomassie staining of the gel. In FIG. 2B, ³²P labeled wild-type or thedouble serine mutant of eNOS (eNOS S635/1179) was affinity purified fromtransfected COS cells and subjected to radiography (upper panel) orWestern blotting (lower panel). The graphical data in FIG. 2B reflectsthe relative amount of labeled protein to the amount of immunoreactiveeNOS in the gel. In FIG. 2C, labeled eNOS was digested with trypsin andpeptides separated by RP-HPLC. The upper chromatogram documents apredominant labeled tryptic peptide that co-migrates with a unlabeledsynthetic phosphopeptide standard (bottom chromatogram). The insetsdemonstrate by linear mode MS of labeled peptide (top) andphosphopeptide standard identical mass ions. In FIG. 2D, recombinantwild-type eNOS or eNOS S1179A were purified and equal amounts (2.4 mg)placed into an in vitro kinase reaction with recombinant Akt asdescribed in Methods. The top panel in FIG. 2D depicts the incorporationof ³²P into eNOS and the bottom panel shows the amount of substrate byCoomassie staining of the gel. The graphical data (n=3) reflects therelative amount of labeled eNOS to the mass of eNOS (Coomassie) in thein vitro kinase reaction.

[0018]FIG. 3. Evidence that serine 1179 is functionally important forAkt stimulated NO release. COS cells were transfected with plasmids forwild-type eNOS or eNOS mutants, in the absence or presence of Akt andthe expression of the proteins and production of NO (assayed as NO₂ ⁻)determined. Interestingly, constructs with the S1179 mutation to A werenot activated by Akt and mutation of S1179 to D resulted in a gain offunction. In A, data are mean±SEM of 4-7 experiments; * representsignificant differences (p<0.05).

[0019]FIGS. 4A-4C. Akt regulates the basal and stimulated production ofNO in endothelial cells. In FIG. 4A, BLMVEC were infected withadenoviral constructs (β-gal as control, myr Akt and AA-Akt) and theamount of NO₂ ⁻ produced over 24 hrs determined (n=3). The inset showsthe expression of eNOS and Akt. In FIG. 4B, lysates from adenoviralinfected BLMVEC were examined for NOS activity. Equal amounts of protein(50 mg) were incubated with various concentrations of free calcium andNOS activity determined (n=3 experiments). In FIG. 4C, BLMVEC wereinfected with adenoviruses as above followed by stimulation with VEGF(40 ng/ml) for 30 min and NO₂ ⁻ release quantified by chemiluminescence.Data are presented as VEGF stimulated release of NO₂ ⁻ after subtractionof basal levels. Data are mean±SEM, n=4; * represent significantdifferences (p<0.05).

[0020]FIGS. 5A and 5B. Purity and dimer/monomer ratio of wild type andeNOS S1179D. In A and B, SDS-PAGE analysis was performed on 7.5%polyacrylamide gels stained with Coomassie Blue. Molecular massstandards (lane 1) and their size in kDa are indicated at the left. Wildtype eNOS (lane 2) and eNOS S1179D (lane 3) (1 μg of each) were resolvedas indicated by arrowheads. In B, proteins (2 μg of each) were resolvedon SDS-PAGE run at 4° C. Molecular mass standards were in lane 1.Nonboiled samples of wild type and eNOS S1179D were resolved in lanes 2and 3, respectively. In lane 4, wild type-eNOS was boiled in SDS samplebuffer.

[0021]FIGS. 6A and 6B. eNOS S1179D has higher rates of NO production (A)and reductase activity (B) than does wild type eNOS. In A, the rate ofNO generated from wild type () and S1179D (◯) eNOS was determined,using the hemoglobin capture assay, as a function of L-arginineconcentration, and data are presented in a double reciprocal plot. In B,DCIP and cytochrome c assays were performed in the presence or absenceof CaM. Values are mean±S.E., n=46 determinations. Similar results wereobtained with at least three enzyme preparations. Significantdifferences (p<0.05) between the wild type and S1179D eNOS are indicatedby the asterisks.

[0022]FIGS. 7A and 7B. NOS activities (A) and NADPH-dependent reductase(B) are increased with eNOS S1179D compared with the wild type enzyme.Hemoglobin capture (A) and NADPH-dependent cytochrome c reduction (B)assays were performed on both wild type and S1179D eNOS. In A, the rateof NO production was determined in the presence of all NOS cofactors(wild type (filled symbols) and S1179D (open symbols) eNOS). The rate ofcytochrome c reduction was performed in the absence of arginine and BH4(A) for wild type (circles) and S1179D (triangles) in the presence(filled symbols) or absence of 120 nM calmodulin (open symbols). Valuesare mean±S.E., n=3-6 determinations from at least three enzymepreparations.

[0023]FIGS. 8A-8D. Calmodulin- and calcium-dependent activation of NOSand reductase activities are slightly enhanced for S1179D eNOS.Calmodulin-dependent hemoglobin capture (A) and cytochrome c reduction(B) were performed on both wild type (filled symbols) and S1179D eNOS(open symbols). The rate of NO production detected by hemoglobin capturemethod is in the presence of all NOS cofactors, whereas cytochrome creduction was performed in the absence of arginine and BH4. In C and D,identical experiments were performed in the presence of increasingconcentrations of free calcium. The insets in C and D depict thecalcium-dependent turnover of S1179D and wild type eNOS in both NOproduction and cytochrome c assays. The maximal turnover rates were asfollows for wild type and S1179D eNOS, respectively: A, 22 and 43 min⁻¹;B, 620 and 1400 min1; C, 58 and 100 min1; and D, 1930 and 3810 min⁻¹.Values are mean±S.E., n=3-6 determinations from at least three enzymepreparations.

[0024]FIGS. 9A and 9B. EGTA-initiated inactivation of NOS is reduced inS1179D eNOS. Hemoglobin capture (A) and reductase assays (B) wereperformed as described earlier, with the following modifications. Thereaction was monitored for 1 min to determine the initial rate; then,EGTA was added to the reaction mixture, and the rate was monitored foran additional 1 min. The free calcium concentration in the reaction was200 μM, and the amount of EGTA added resulted in final concentrations of0, 200, 400, and 600 μM chelator. The specific activities are normalizedto 100% for wild type and S1179D eNOS. Values are mean±S.E., n=3-6determinations from at least three enzyme preparations. nd, nodetectable activity for wild type eNOS.

DETAILED DESCRIPTION OF THE INVENTION A. General Description

[0025] The present inventions are based, in part, upon the discoverythat the serine/threonine protein kinase, Akt (protein kinase B), candirectly phosphorylate eNOS on serine 1179 (serine 1177 in human eNOS),and activate the enzyme leading to NO production while mutant eNOS(S1179A) is resistant to Akt phosphorylation and activation. Moreover,using adenoviral mediated gene transfer activated Akt increases basal NOrelease from endothelial cells and activation deficient Akt attenuatesVEGF stimulated NO production. Thus, eNOS is a newly described Aktsubstrate linking signal transduction via Akt to the release of thegaseous second messenger, NO. The present inventions are also based inpart on the findings that mutant eNOS, for instance, S1179D, exhibits anincrease in the rate of NO production and an increase in reductaseactivity.

[0026] The demonstration that NO production is regulated by Aktdependent phosphorylation of eNOS provides novel constitutively activeeNOS mutants for use in gene therapy aimed at improving endothelialfunction in cardiovascular diseases associated with dysfunction in thesynthesis or biological activity of NO. Such diseases include postangioplasty restenosis, hypertension, atherosclerosis, heart failureincluding myocardial infarction, diabetes, and diseases with defectiveangiogenesis. This discovery also provides a novel therapeutic targetuseful for the design of drugs useful for treating diseases associatedwith dysfunction in the synthesis or biological activity of NO.

[0027] The present invention also provides novel constitutively activenNOS mutants which have a substituted amino acid corresponding toresidue 1412 of rat nNOS or 1415 or human nNOS for use in gene therapyaimed at the treatment of diseases.

B. Specific Embodiments

[0028] Production of NOS Mutant Proteins or Polypeptides

[0029] The present invention provides NOS proteins or polypeptides,allelic variants of NOS proteins, and conservative amino acidsubstitutions of NOS proteins, all of which contain a mutation of aserine residue which is the site of Akt mediated phosphorylation. Forinstance, the proteins or polypeptides of the invention include but arenot limited to: (1) human eNOS proteins which comprise a mutation ofresidue 1177 (Janssens et al. (1992) J. Biol. Chem. 267:14519-14522which is herein incorporated by reference in its entirety) from a serineto another amino acid, such as alanine, and are resistant to Aktmediated phosphorylation; (2) bovine eNOS proteins which comprise amutation of residue 1179 (SEQ ID NO: 2 of U.S. Pat. No. 5,498,539, whichis herein incorporated by reference in its entirety) from a serine toanother amino acid, such as alanine, and are resistant to Akt mediatedphosphorylation; (3) human nNOS proteins which comprise a mutation ofresidue 1415 from a serine to another amino acid, such as alanine, andare resistant to Akt mediated phosphorylation; (4) rat nNOS proteinswhich comprise a mutation of residue 1412 from a serine to another aminoacid, such as alanine, and are resistant to Akt mediatedphosphorylation; (5) human eNOS proteins which comprise a mutation ofresidue 1177 from a serine to an amino acid containing a negativelycharged R group, such as aspartic or glutamic acid, and areconstitutively active and exhibit increased NO production and increasedreductase activity; (6) bovine eNOS proteins which comprise a mutationof residue 1179 from a serine to an amino acid containing a negativelycharged R group, such as aspartic or glutamic acid, and areconstitutively active and exhibit increased NO production and increasedreductase activity; (7) human nNOS proteins which comprise a mutation ofresidue 1415 from a serine to an amino acid containing a negativelycharged R group, such as aspartic or glutamic acid, and areconstitutively active and exhibit increased NO production and increasedreductase activity; (8) rat nNOS proteins which comprise a mutation ofresidue 1412 from a serine to an amino acid containing a negativelycharged R group, such as aspartic or glutamic acid, and areconstitutively active and exhibit increased NO production and increasedreductase activity; and (9) NOS proteins from species other than humans,cows, or rat which are modified to contain an amino acid other thanserine at a position corresponding to the serine at position 1177 in thehuman eNOS or position 1179 in the bovine eNOS, position 1412 in the ratnNOS, and position 1415 in the human nNOS and which are either resistantto Akt phosphorylation, are constitutively active, or exhibit increasedNO production and increased reductase activity. NOS mutants may also beproduced by mutating other amino acids in the phosphorylation motifRXRXXS/T.

[0030] The present invention provides constitutively active NOSpolypeptides, preferably eNOS or nNOS, exhibiting increased NOproduction and reductase activity and comprising a mutation at theserine residue at the site of Akt mediated phosphorylation. It is alsowithin the skill of the artisan to obtain conservative variants such assubstitutions, deletions, and insertions mutants of these NOSpolypeptides exhibiting increased NO production and reductase activity.As used herein, a conservative variant refers to alterations in theamino acid sequence that do not adversely affect the ability ofconstitutively active NOS, preferably eNOS or nNOS, to produce NO or thereductase activity of constitutively active NOS, preferably eNOS ornNOS. A substitution, insertion, or deletion is said to adversely affectconstitutively active NOS polypeptide, when the altered sequence affectsthe ability of constitutive NOS, such that it does not produce NO at anincreased level and does not have increased reductase activity ascompared to the wild-type NOS. For example, the overall charge,structure or hydrophobic/hydrophilic properties of constitutive NOS canbe altered without adversely affecting the activity of constitutive NOS.Accordingly, the amino acid sequence of NOS polypeptide can be altered,for instance to render the polypeptide more hydrophobic or hydrophilic,without adversely affecting the activity of NOS.

[0031] As used herein, a “constitutively active” mutant or variant ofNOS, whether modified or isolated from a natural source, refers to a NOSprotein, preferably an eNOS or a nNOS, which produces NO at a ratehigher than native NOS containing a serine in its unphosphorylated format an amino acid residue corresponding to residue 1177 in human NOS orresidue 1179 in bovine NOS. Preferred constitutively active variantscomprise an amino acid with a negatively charged R group, such asaspartic or glutamic acid, at the amino acid residue corresponding tothe serine at position 1177 in the human eNOS or position 1179 in thebovine NOS.

[0032] The present invention provides NOS proteins or polypeptides,allelic variants of NOS proteins, and conservative amino acidsubstitutions of NOS proteins that contain a substituted amino acidresidue corresponding to residue 1177 of bovine eNOS, to residue 1179 ofhuman eNOS, to residue 1412 of rat nNOS, and to residue 1415 of humannNOS, wherein the substituted amino acid residue comprises anon-negatively charged R group, such as alanine.

[0033] The NOS proteins, preferably eNOS or nNOS proteins, of thepresent invention may be in isolated form. As used herein, a protein issaid to be isolated when physical, mechanical or chemical methods areemployed to remove the protein from cellular constituents that arenormally associated with the protein. A skilled artisan can readilyemploy standard purification methods to obtain an isolated protein.

[0034] Also included in the invention are NOS peptides which span theAkt phosphorylation site corresponding to residue 1179 in bovine eNOS,residue 1177 in human eNOS, residue 1412 in rat nNOS or residue 1415 inhuman nNOS. Peptides may contain a serine at the phosphorylation siteor, preferably, may contain a substitution of the serine at positioncorresponding to residue 1179 in bovine eNOS, residue 1177 in humaneNOS, residue 1412 in rat nNOS, or residue 1415 in human nNOS. Suchsubstitutions include, but are not limited to, amino acids with an Rgroup that mimics serine in its phosphorylated state, such as asparticacid or glutamic acid. Such substitutions also include, amino acids witha non-negative R group, such as alanine. Peptides spanning this site maybe about 3, 5, 7, 10, 12, 15, 17, 20, 25, 30, 40, 50 or more amino acidsin length.

[0035] NOS proteins, polypeptides or peptides of the invention may beprepared by any means available, including recombinant expression froman NOS cDNA which has been modified to replace or alter the nucleotidetriplet encoding a serine corresponding to the serine at position 1177in the human eNOS, position 1179 in the bovine eNOS, residue 1412 in ratnNOS, or residue 1415 in human nNOS. Any available technique may be usedto mutate the nucleotide triplet encoding the serine residue, such ashomologous recombination, site-directed mutagenesis or PCR mutagenesis(see, Sambrook et al., Molecular Cloning, Cold Spring Harbor LaboratoryPress, 1989). Starting cDNAs may include the human and bovine eNOS cDNAsas well as cDNAs encoding eNOS proteins of other animal species,including but not limited to rabbit, rat, murine, porcine, ovine, equineand non-human primate species.

[0036] As used herein, a nucleic acid molecule encoding a NOS protein orpolypeptide, preferably eNOS or nNOS protein or polypeptide, of theinvention is said to be “isolated” when the nucleic acid molecule issubstantially separated from contaminant nucleic acid encoding otherpolypeptides from the source of nucleic acid.

[0037] The present invention further provides fragments of the encodingnucleic acid molecule. As used herein, a fragment of an encoding nucleicacid molecule refers to a small portion of the entire protein encodingsequence. The size of the fragment will be determined by the intendeduse. For example, if the fragment is chosen so as to encode an activeportion of the protein, the fragment will need to be large enough toencode the functional region(s) of the protein, including the Aktphosphorylation site. If the fragment is to be used as a nucleic acidprobe or PCR primer, then the fragment length is chosen so as to obtaina relatively small number of false positives during probing/priming to aregion which spans or flanks the NOS Akt phosphorylation site.

[0038] Fragments of the encoding nucleic acid molecules of the presentinvention (i.e., synthetic oligonucleotides) that are used as probes orspecific primers for the polymerase chain reaction (PCR), or tosynthesize gene sequences encoding proteins of the invention can easilybe synthesized by chemical techniques, for example, the phosphotriestermethod of Matteucci, et al. 1981 J. Am. Chem. Soc. 103:3185-3191) orusing synthesis methods. In addition, larger DNA segments can readily beprepared by well known methods, such as synthesis of a group ofoligonucleotides that define various modular segments of the gene,followed by ligation of oligonucleotides to build the complete modifiedgene.

[0039] The encoding nucleic acid molecules of the present invention mayfurther be modified so as to contain a detectable label for diagnosticand probe purposes. A variety of such labels are known in the art andcan readily be employed with the encoding molecules herein described.Suitable labels include, but are not limited to, biotin, radiolabelednucleotides and the like. A skilled artisan can employ any of the artknown labels to obtain a labeled encoding nucleic acid molecule.

[0040] The present invention further provides recombinant DNA molecules(rDNAs) that contain an NOS coding sequence as described above. As usedherein, a rDNA molecule is a DNA molecule that has been subjected tomolecular manipulation. Methods for generating rDNA molecules are wellknown in the art, for example, see Sambrook et al., Molecular Cloning(1989). In the preferred rDNA molecules, a coding DNA sequence isoperably linked to expression control sequences and/or vector sequences.

[0041] The choice of vector and/or expression control sequences to whichone of the protein family encoding sequences of the present invention isoperably linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., protein expression, and the hostcell to be transformed. A vector contemplated by the present inventionis at least capable of directing the replication or insertion into thehost chromosome, and preferably also expression, of the structural geneincluded in the rDNA molecule.

[0042] Expression control elements that are used for regulating theexpression of an operably linked protein encoding sequence are known inthe art and include, but are not limited to, inducible promoters,constitutive promoters, secretion signals, and other regulatoryelements. Preferably, the inducible promoter is readily controlled, suchas being responsive to a nutrient in the host cell's medium.

[0043] In one embodiment, the vector containing a coding nucleic acidmolecule will include a prokaryotic replicon, i.e., a DNA sequencehaving the ability to direct autonomous replication and maintenance ofthe recombinant DNA molecule extrachromosomally in a prokaryotic hostcell, such as a bacterial host cell transformed therewith. Suchreplicons are well known in the art. In addition, vectors that include aprokaryotic replicon may also include a gene whose expression confers adetectable marker such as a drug resistance. Typical bacterial drugresistance genes are those that confer resistance to ampicillin ortetracycline.

[0044] Vectors that include a prokaryotic replicon can further include aprokaryotic or bacteriophage promoter capable of directing theexpression (transcription and translation) of the coding gene sequencesin a bacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding of RNApolymerase and transcription to occur. Promoter sequences compatiblewith bacterial hosts are typically provided in plasmid vectorscontaining convenient restriction sites for insertion of a DNA segmentof the present invention. Typical of such vector plasmids are pUC8,pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond,Calif.), pPL and pKK223 available from Pharmacia, Piscataway, N.J.

[0045] Expression vectors compatible with eukaryotic cells, preferablythose compatible with vertebrate cells, can also be used to form a rDNAmolecules that contains a coding sequence. Eukaryotic cell expressionvectors are well known in the art and are available from severalcommercial sources. Typically, such vectors are provided containingconvenient restriction sites for insertion of the desired DNA segmentTypical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d(International Biotechnologies, Inc.), pTDT1 (ATCC, #31255), and thelike eukaryotic expression vectors.

[0046] Eukaryotic cell expression vectors used to construct the rDNAmolecules of the present invention may further include a selectablemarker that is effective in an eukaryotic cell, preferably a drugresistance selection marker. A preferred drug resistance marker is thegene whose expression results in neomycin resistance, i.e., the neomycinphosphotransferase (neo) gene. (Southern et al. (1982) J. Mol. Anal.Genet. 1:327-341) Alternatively, the selectable marker can be present ona separate plasmid, and the two vectors are introduced byco-transfection of the host cell and selected by culturing theappropriate drug for the selectable marker.

[0047] The present invention further provides host cells transformed ortransfected with a nucleic acid molecule that encodes an NOS protein,preferably eNOS or nNOS protein, of the present invention. The host cellcan be either prokaryotic or eukaryotic. Eukaryotic cells useful forexpression of a protein of the invention are not limited, so long as thecell line is compatible with cell culture methods and compatible withthe propagation of the expression vector and expression of the geneproduct. Preferred eukaryotic host cells include, but are not limitedto, yeast, insect and mammalian cells, preferably vertebrate cells suchas those from a mouse, rat, monkey or human cell line. Preferredeukaryotic host cells include Chinese hamster ovary (CHO) cellsavailable from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3available from the ATCC as CRL 1658, baby hamster kidney cells (BHK),and the like eukaryotic tissue culture cell lines. Any prokaryotic hostcan be used to express a rDNA molecule encoding a protein of theinvention. The preferred prokaryotic host is E. coli, particularly forthe constitutively active NOS mutants.

[0048] Transformation or transfection of appropriate cell hosts with arDNA molecule of the present invention is accomplished by well knownmethods that typically depend on the type of vector used and host systememployed. With regard to transformation of prokaryotic host cells,electroporation and salt treatment methods are typically employed, see,for example, Cohen et al., (1972) Proc. Natl. Acad. Sci. USA 69:2110;and Maniatis et al., Molecular Cloning, A Laboratory Mammal, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1982). With regard totransformation of vertebrate cells with vectors containing rDNAs,electroporation, cationic lipid or salt treatment methods are typicallyemployed, see, for example, Graham et al. 1983) Virol. 52:456; Wigler etal., (1979) Proc. Natl. Acad. Sci. USA 76:1373-76. Successfullytransformed or transfected cells, i.e., cells that contain a rDNAmolecule of the present invention, can be identified by well knowntechniques including the selection for a selectable marker. For example,cells resulting from the introduction of an rDNA of the presentinvention can be cloned to produce single colonies. Cells from thosecolonies can be harvested, lysed and their DNA content examined for thepresence of the rDNA using a method such as that described by Southern(1975) J. Mol. Biol. 98:503 or Berent et al. (1985) Biotech. 3:208, orthe proteins produced from the cell assayed via an immunological method.

[0049] The present invention further provides methods for producing aNOS protein, preferably an eNOS protein or a nNOS protein, of theinvention using nucleic acid molecules herein described. In generalterms, the production of a recombinant form of a protein typicallyinvolves the following steps. A nucleic acid molecule is first obtainedthat encodes a protein of the invention. If the encoding sequence isuninterrupted by introns, it is directly suitable for expression in anyhost. The nucleic acid molecule is then preferably placed in operablelinkage with suitable control sequences, as described above, to form anexpression unit containing the protein open reading frame. Theexpression unit is used to transform or transfect a suitable host andthe transformed or transfected host is cultured under conditions thatallow the production of the recombinant protein. Optionally therecombinant protein is isolated from the medium or from the cells;recovery and purification of the protein may not be necessary in someinstances where some impurities may be tolerated.

[0050] Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in appropriate hosts. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using appropriate replicons and control sequences, as setforth above. The control sequences, expression vectors, andtransformation or transfection methods are dependent on the type of hostcell used to express the gene and were discussed in detail earlier.Suitable restriction sites can, if not normally available, be added tothe ends of the coding sequence so as to provide an excisable gene toinsert into these vectors. A skilled artisan can readily adapt anyhost/expression system known in the art for use with the nucleic acidmolecules of the invention to produce recombinant protein.

[0051] Gene Therapy

[0052] Any appropriate gene delivery system combined with a suitablegene expression system using the most appropriate route of delivery isencompassed by the present invention. For instance, NOS mutant orvariant genes, preferably eNOS or nNOS mutant or variant genes, of theinvention or Akt genes may be transferred to the heart (or skeletalmuscle), including cardiac myocytes (and skeletal myocytes), in vitro orin vivo to direct production of the encoded protein. Particularly usefulare human Akt genes and NOS mutants, preferably human eNOS, containingan amino acid with a negatively charged R group, such as aspartic orglutamic acid, at a position corresponding to serine 1177 in human eNOS.Routes of administering NOS mutant or variant genes include, but are notlimited to, intravascular, intramuscular, intraperitoneal, intradermal,and intraarterial injection.

[0053] The adenovirus gene delivery system offers several advantages:adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of cell types; and (iv)be used with a large number of available vectors containing differentpromoters. Also, because adenoviruses are stable in the bloodstream,they can be administered by intravenous injection. A preferred deliveryvector is a helper-independent replication deficient human adenovirus 5,although other delivery means are available and may be used, includingdelivery of nucleic acids directly to the cells of interest (see Sawa etal. (1998) Gene Ther. 5(11):1472-80; Labhasetwar et al. (1998) J. Pharm.Sci. 87(11):1347-50; Lin et al. (1997) Hypertension 30:307-313; Chen etal. (1997) Circ. Res. 80(3):327-335; Channon et al. (1996) Cardiovasc.Res. 32:962-972; Harv Heart Lett. (1999) 9(8):5-6; and Nabel et al.(1999) Nat. Med. 5(2):141-2.

[0054] Using the adenovirus 5 system, transfection frequencies ofgreater than 60% have been demonstrated in myocardial cells in vivo by asingle intracoronary injection (Giordano and Hammond (1994) Clin. Res.42: 123A). Non-replicative recombinant adenoviral vectors areparticularly useful in transfecting coronary endothelium and cardiacmyocytes resulting in highly efficient transfection after intracoronaryinjection. Non-replicative recombinant adenoviral vectors are alsouseful for transfecting desired cells of the peripheral vascular system(see U.S. Pat. No. 5,792,453, which is herein incorporated by referencein its entirety).

[0055] Adenoviral vectors used in the present invention can beconstructed by the rescue recombination technique described in Graham etal. (1988) Virology 163:614-617. Briefly, the eNOS transgene is clonedinto a shuttle vector that contains a promoter, polylinker and partialflanking adenoviral sequences from which E1A/E1B genes have beendeleted. As the shuttle vector, plasmid pAC1 (Virology 163:614-617,1988) (or an analog) which encodes portions of the left end of the humanadenovirus 5 genome (Virology 163:614-617, 1988) minus the early proteinencoding E1A and E1B sequences that are essential for viral replication,and plasmid ACCMVPLPA (J. Biol. Chem. 267:25129-25134, 1992) whichcontains polylinker, the CMV promoter and SV40 polyadenylation signalflanked by partial adenoviral sequences from which the EA/E1B genes havebeen deleted can be exemplified. The use of plasmid PAC1 or ACCMVPLAfacilitates the cloning process. The shuttle vector is thenco-transfected with a plasmid which contains the entire human adenoviral5 genome with a length too large to be encapsidated, into 293 cells.Co-transfection can be conducted by calcium phosphate precipitation orlipofection (Biotechniques 15:868-872, 1993). Plasmid JM17 encodes theentire human adenovirus 5 genome plus portions of the vector pBR322including the gene for ampicillin resistance (4.3 kb). Although JM17encodes all of the adenoviral proteins necessary to make mature viralparticles, it is too large to be encapsidated (40 kb versus 36 kb forwild type). In a small subset of co-transfected cells, rescuerecombination between the transgene containing the shuttle vector suchas plasmid pAC1 and the plasmid having the entire adenoviral 5 genomesuch as plasmid pJM17 provides a recombinant genome that is deficient inthe E1A/E1B sequences, and that contains the transgene of interest butsecondarily loses the additional sequence such as the pBR322 sequencesduring recombination, thereby being small enough to be encapsidated. TheCMV driven beta-galactosidase encoding adenovirus HCMVSP1lacZ (Clin.Res. 42:123A, 1994) can be used to evaluate efficiency of gene transferusing X-gal treatment.

[0056] In another embodiment, the gene encoding NOS, preferably eNOS ornNOS, may be introduced in vivo via an attenuated or defective DNAvirus, such as but not limited to herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, andadeno-associated virus (AAV). Defective viruses, which entirely oralmost entirely lack viral genes, are preferred. Defective virus is notinfective after introduction into a cell. Use of defective, vitalvectors allows for administration to cells in a specific, localizedarea, without concern that the vector can infect other cells. Thus, aparticular locus, e.g., in the brain or spinal chord, can bespecifically targeted with the vector. In a specific embodiment, adefective herpes virus 1 (HSV1) vector may be used (Kaplitt et al.(1991) Molec. Cell. Neurosci. 2:320-330). In yet another embodiment, theviral vector is an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al. (J. Clin. Invest. 90:626-630(1992)). In a yet a further embodiment, the vector is a defectiveadeno-associated virus vector (Samulski et al. (1987) J. Virol.61:3096-3101; Samulski et al. (1989) J. Virol. 63:3822-3828).

[0057] The present invention also contemplates the use of cell targetingnot only by delivery of the transgene into the coronary artery, orfemoral artery, for example, but also the use of tissue-specificpromoters. By fusing, for example, tissue-specific transcriptionalcontrol sequences of left ventricular myosin light chain-2 (MLC[2V]) ormyosin heavy chain (MHC) to a transgene such as the NOS genes of theinvention within the adenoviral construct, transgene expression islimited to ventricular cardiac myocytes. The efficacy of gene expressionand degree of specificity provided by MLC[2V ] and MHC promoters withlacZ have been determined, using the recombinant adenoviral system ofthe present invention. Cardiac-specific expression has been reportedpreviously by Lee et al. (J. Biol. Chem. 267:15875-15885 (1992)). TheMLC[2V] promoter is comprised of 250 bp, and fits easily within theadenoviral-5 packaging constraints. The myosin heavy chain promoter,known to be a vigorous promoter of transcription, provides a reasonablealternative cardiac-specific promoter and is comprised of less than 300bp. Smooth muscle cell promoters such as SM22 alpha promoter (Kemp etal., (1995) Biochem J 310 (Pt 3):1037-43) and SM alpha actin promoter(Shimizu et al. (1995) J Biol Chem 270(13):763-143) are also available.Other promoters, such as the troponin-C promoter, while highlyefficacious and sufficiently small, lacks adequate tissue specificity.By using the MLC[2V ] or MHC promoters and delivering the transgene invivo, it is believed that the cardiac myocyte alone (that is withoutconcomitant expression in endothelial cells, smooth muscle cells, andfibroblasts within the heart) will provide adequate expression of theNOS protein.

[0058] Limiting expression to the cardiac myocyte also has advantagesregarding the utility of gene transfer for the treatment of clinicalmyocardial ischemia. By limiting expression to the heart, one avoids thepotentially harmful effect of angiogenesis in non-cardiac tissues suchas the retina. In addition, of the cells in the heart, the myocyte wouldlikely provide the longest transgene expression since the cells do notundergo rapid turnover; expression would not therefore be decreased bycell division and death as would occur with endothelial cells.Endothelial-specific promoters are already available for this purpose.Examples of endothelial specific promoters include the Tie-2 promoter(Schlaeger et al. (1997) Proc Natl Acad Sci 1; 94(7):3058-63), theendothelin promoter (Lee et al. (1990) J. Biol. Chem. 265:10446-10450),and the eNOS promoter (Zhang et al. (1995) J Biol. Chem270(25):15320-6).

[0059] The present invention includes, with regard to the treatment ofheart disease, targeting the heart by intracoronary or intramuscularinjection with a high titer of the vector and transfecting all celltypes is presently preferred. Diseases such as erectile dysfunction andcardiovascular diseases including, myocardial infarction, myocardialischemia, heart failure, restenosis, stent stenosis, post-angioplastystenosis, and by-pass graft failure may be treated as described usingthe NOS transgenes, preferably eNOS or nNOS transgenes.

[0060] Successful recombinant vectors can be plaque purified accordingto standard methods. The resulting viral vectors are propagated on 293cells which provide E1A and E1B functions to in trans to titers in thepreferred about 10¹⁰-about 10¹² viral particles/ml range. Cells can beinfected at 80% confluence and harvested 48 hours later. After 3freeze-thaw cycles the cellular debris is pelleted by centrifugation andthe virus purified by CsCl gradient ultracentrifugation (double CsClgradient ultracentrifugation is preferred). Prior to in vivo injection,the viral stocks are desalted by gel filtration through Sepharosecolumns such as G25 Sephadex. The product is then filtered through a 30micron filter, thereby reducing deleterious effects of intracoronaryinjection of unfiltered virus (life threatening cardiac arrhythmias) andpromoting efficient gene transfer. The resulting viral stock has a finalviral titer in the range of 10¹⁰-10¹² viral particles/ml. Therecombinant adenovirus must be highly purified, with no wild-type(potentially replicative) virus. Impure constructs can cause an intenseimmune response in the host animal. From this point of view, propagationand purification may be conducted to exclude contaminants and wild-typevirus by, for example, identifying successful recombinants with PCRusing appropriate primers, conducting two rounds of plaque purification,and double CsCl gradient ultracentrifugation. Additionally, the problemsassociated with cardiac arrhythmias induced by adenovirus vectorinjection into patients can be avoided by filtration of the recombinantadenovirus through an appropriately-sized filter prior to intracoronaryinjection. This strategy also appears to substantially improve genetransfer and expression.

[0061] The viral stock can be in the form of an injectable preparationcontaining pharmaceutically acceptable carrier such as saline, forexample, as necessary. The final titer of the vector in the injectablepreparation is preferably in the range of about 10⁷-about 10¹³ viralparticles which allows for effective gene transfer. Other pharmaceuticalcarriers, formulations and dosages are described below. The adenovirustransgene constructs are delivered to the myocardium by directintracoronary (or graft vessel) injection using standard percutaneouscatheter based methods under fluoroscopic guidance, at an amountsufficient for the transgene to be expressed to a degree which allowsfor highly effective therapy. The injection may be made deeply into thelumen (about 1 cm within the arterial lumen) of the coronary arteries(or graft vessel), and preferably be made in both coronary arteries, asthe growth of collateral blood vessels is highly variable withinindividual patients. By injecting the material directly into the lumenof the coronary artery by coronary catheters, it is possible to targetthe gene rather effectively, and to minimize loss of the recombinantvectors to the proximal aorta during injection. It is known that geneexpression when delivered in this manner does not occur in hepatocytesand viral RNA cannot be found in the urine at any time afterintracoronary injection. Any variety of coronary catheter, or a Stackperfusion catheter, for example, can be used in the present invention.In addition, other techniques known to those having ordinary skill inthe art can be used for transfer of NOS genes, preferably eNOS or nNOS,to the arterial wall.

[0062] For the treatment of peripheral vascular disease, a diseasecharacterized by insufficient blood supply to the legs, recombinantadenovirus expressing a NOS, preferably an eNOS or a nNOS, peptide orprotein of the invention may be delivered by a catheter inserted intothe proximal portion of the femoral artery or arteries, therebyeffecting gene transfer into the cells of the skeletal muscles receivingblood flow from the femoral arteries.

[0063] In instances wherein a transgene or nucleic acid encoding an NOS,preferably an eNOS or a nNOS, or Akt protein of the invention is firsttransferred to endothelial or vascular smooth muscle cells in vitro,including the patients own cells, DNA may be transfected into the cellsdirectly (see U.S. Pat. No. 5,658,565). Generally, to transfect targetcells, a plasmid vector comprising a DNA sequence encoding an Akt or NOSof the invention or a biologically active fragment thereof may beutilized in liposome-mediated transfection of the target cell. Thestability of liposomes, coupled with the impermeable nature of thesevesicles, makes them useful vehicles for the delivery of therapeutic DNAsequences (for a review, see Mannino and Gould-Forgerite (1988)BioTechniques 6(7): 682-690). Liposomes are known to be absorbed by manycell types by fusion. In one embodiment, a cationic liposome containingcationic cholesterol derivatives, such as SF-chol or DC-chol, may beutilized. The DC-chol molecule includes a tertiary amino group, a mediumlength spacer arm and a carbamoyl linker bond as described by Gao andHuang (Biochem. Biophys. Res. Comm. 179: 280-285, 1991).

[0064] In another embodiment regarding the use of liposome technology,the viral or nonviral based vector comprising the DNA sequence encodinga biologically active NOS protein fragment, preferably eNOS or nNOSprotein fragment, is delivered to the target cell by transfection of thetarget cell with lipofectamine (Bethesda Research Laboratory).Lipofectamine is a 3:1 Liposome formulation of the polycationic lipid2,3dioleyloxy-N-[2(sperminecarboxymido)ethyl]-N,N-dimethyl-1-propanaminiumtricfluoroacetate (DOPSA) and the neutral lipiddioleoly-phosphatidylethanolamine (DOPE).

[0065] Other non-viral modes of gene delivery include, but are notlimited to: (a) direct injection of naked DNA; (b) calcium phosphate[Ca₃(PO₄)₂] mediated cell transfection; (c) mammalian host celltransfection by electroporation; (d) DEAE-dextran mediated celltransfection; (e) polybrene mediated delivery; (f) protoplast fusion;(g) microinjection; and (h) polylysine mediated transformation, with thegenetically engineered cells transferred back to the mammalian host.

[0066] Production of Transgenic Animals

[0067] Transgenic animals containing a mutant NOS gene, preferably amutant eNOS or nNOS gene, as described herein are also included in theinvention. Transgenic animals are genetically modified animals intowhich recombinant, exogenous or cloned genetic material has beenexperimentally transferred. Such genetic material is often referred toas a “transgene”. The nucleic acid sequence of the transgene, in thiscase a form of NOS, may be integrated either at a locus of a genomewhere that particular nucleic acid sequence is not otherwise normallyfound or at the normal locus for the transgene. The transgene mayconsist of nucleic acid sequences derived from the genome of the samespecies or of a different species than the species of the target animal.

[0068] The term “germ cell line transgenic animal” refers to atransgenic animal in which the genetic alteration or genetic informationwas introduced into a germ line cell, thereby conferring the ability ofthe transgenic animal to transfer the genetic information to offspring.If such offspring in fact possess some or all of that alteration orgenetic information, then they too are transgenic animals.

[0069] The alteration or genetic information may be foreign to thespecies of animal to which the recipient belongs, foreign only to theparticular individual recipient, or may be genetic information alreadypossessed by the recipient. In the last case, the altered or introducedgene may be expressed differently than the native gene.

[0070] Transgenic animals can be produced by a variety of differentmethods including transfection, electroporation, microinjection, genetargeting embryonic stem cells and recombinant viral and retroviralinfection (see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,602,307;Mullins et al. (1993) Hypertension 22(4):630-633; Brenin et al. (1997)Surg. Oncol. 6(2)99-110; Tuan (ed.), Recombinant Gene ExpressionProtocols, Methods in Molecular Biology No. 62, Humana Press (1997)).

[0071] A number of recombinant or transgenic mice have been produced,including those which express an activated oncogene sequence (U.S. Pat.No. 4,736,866); express simian SV 40 T-antigen (U.S. Pat. No.5,728,915); lack the expression of interferon regulatory factor 1(IRF-1) (U.S. Pat. No. 5,731,490); exhibit dopaminergic dysfunction(U.S. Pat. No. 5,723,719); express at least one human gene whichparticipates in blood pressure control (U.S. Pat. No. 5,731,489);display greater similarity to the conditions existing in naturallyoccurring Alzheimer's disease (U.S. Pat. No. 5,720,936); have a reducedcapacity to mediate cellular adhesion (U.S. Pat. No. 5,602,307); possessa bovine growth hormone gene (Clutter et al. (1996) Genetics143(4):1753-1760); or, are capable of generating a fully human antibodyresponse (McCarthy (1997) The Lancet 349(9049):405).

[0072] While mice and rats remain the animals of choice for mosttransgenic experimentation, in some instances it is preferable or evennecessary to use alternative animal species. Transgenic procedures havebeen successfully utilized in a variety of non-murine animals, includingsheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits,cows and guinea pigs (see e.g., Kim et al. (1997) Mol. Reprod. Dev.46(4):515-526; Houdebine (1995) Reprod. Nutr. Dev. 35(6):609-617;Petters (1994) Reprod. Fertil. Dev. 6(5):643-645; Schnieke et al. (1997)Science 278(5346):2130-2133; and Amoah (1997) J. Animal Science75(2):578-585).

[0073] The method of introduction of nucleic acid fragments intorecombination competent mammalian cells can be by any method whichfavors co-transformation of multiple nucleic acid molecules. Detailedprocedures for producing transgenic animals are readily available to oneskilled in the art, including the disclosures in U.S. Pat. No. 5,489,743and U.S. Pat. No. 5,602,307. Furthermore, the production of NOStransgenic animals is well developed. For instance, transgenic micewhich inducibly express or overexpress wild type eNOS have been produced(see Ohashi et al. (1998) J. Clin. Invest. 102(12):2061-71; and Drummondet al. (1998) J. Clin. Invest. 102(12):2033-4). These methods may beused to produce transgenic mice which express the NOS mutants of theinvention.

[0074] Therapeutic Screening Assays

[0075] The discovery that phosphorylation of eNOS regulates its activityallows for the development of screening assays to identify agents whichmodulate Akt regulated NOS, preferably eNOS or nNOS, activity orexpression. Any available format may be used, including in vivotransgenic animal assays, in vitro protein based assays, cell cultureassays and high-throughput formats.

[0076] In many drug screening programs which test libraries ofcompounds, high throughput assays are desirable in order to maximize thenumber of compounds surveyed in a given period of time. Assays which areperformed in cell-free systems, such as may be derived with purified orsemi-purified proteins, are often preferred as “primary” screens in thatthey can be generated to permit rapid development and relatively easydetection of an alteration in a molecular target which is mediated by atest compound. Moreover, the effects of cellular toxicity and/orbioavailability of the test compound can be generally ignored in the invitro system, the assay instead being focused primarily on the effect ofthe drug on the molecular target as may be manifest in an inhibition of,for instance, binding between molecules.

[0077] Cell or tissue culture based assays may be performed, forexample, by plating COS-7 cells (100 mm dish) and transfecting with NOS(7.5-30 mg) and Akt (1 mg) plasmids using calcium phosphate. To balanceall transfections, an expression vector for β-galactosidase cDNA may becotransfected. Twenty-four to forty-eight hours after transfection, theexpression of appropriate proteins (40-80 mg) may be confirmed byWestern blot analysis using NOS mAb (9D10, Zymed), HA mAb (12CA5,Boehringer Mannheim), iNOS pAb (Zymed Laboratories) or nNOS mAb (ZymedLaboratories).

[0078] Twenty-four to forty-eight hours after transfection, media may beprocessed for the measurement of nitrite (NO₂ ⁻), the stable breakdownproduct of NO in aqueous solution, by NO specific chemiluminescence asdescribed (Sessa et al., 1995). Media is deproteinized and samplescontaining NO₂ ⁻ are refluxed in glacial acetic acid containing sodiumiodide. Under these conditions, NO₂ ⁻ is quantitatively reduced to NOwhich is quantified by a chemiluminescence detector after reaction withozone in a NO analyzer (Sievers, Boulders, Colo.). In all experiments,controls may be prepared by inhibiting NO₂ ⁻ release by the use of a NOSinhibitor. In addition, NO₂ ⁻-release from cells transfected with theβ-galactosidase cDNA may subtracted to control for background levels ofNO₂ ⁻ found in serum or media. cGMP accumulation in COS may also be usedas a bioassay for the production of NO as described. In an alternativeformat, the conversion of ³H-L-arginine to ³H-L-citrulline may be usedto determined NOS activity in COS cell or endothelial cell lysates aspreviously described (Garcia-Cardena et al., 1998).

[0079] For in vivo phosphorylation studies, COS cells may be transfectedwith the cDNAs for wild-type or S635 (control), bovine 1179A, D, or EeNOS, human 1177 D or E eNOS, rat 1412D or E nNOS, human 1415D or E nNOSand HA-Akt overnight. 36 hrs after transfection, cells are placed intodialyzed serum replete, phosphate-free Dulbecco's minimum essentialmedium supplemented with 80 μCi/ml of ³²P orthophosphoric acid for 3 hr.A cell aliquot may be pretreated with wortmannin (500 nM) in thephosphate-free media for 1 hr and during the labeling. Lysates are thenharvested, NOS solubilized and partially purified by ADP sepharoseaffinity chromatography as previously described and the ³²Pincorporation into NOS visualized after SDS-PAGE (7.5%) byautoradiography and the amount of NOS protein verified by Westernblotting for NOS.

[0080] For in vitro phosphorylation studies, recombinant NOS purifiedfrom E. coli, eNOS purified from another source, or NOS peptidesspanning the Akt phosphorylation site are incubated with wild-type orkinase inactive Akt immunoprecipitated from transfected COS cells.Briefly, the NOS proteins or peptides are incubated with ³²P g-ATP (2ml, specific activity 3000 Ci/mmol), ATP (50 mM), DTT (1 mM), in abuffer containing HEPES (20 mM, pH=7.4), MnCl₂ (10 mM), MgCl₂ (10 mM)and immunoprecipitated Akt for 20 min at room temperature.

[0081] In experiments examining the in vitro phosphorylation ofwild-type and mutant NOS, recombinant Akt (1 mg) purified frombaculovirus infected SF9 cells, is incubated with wild-type, S1179Abovine eNOS, S1177A human eNOS, S1179 D or E bovine eNOS, S1177D or Ehuman eNOS, S1412D or E rat nNOS, or S1415D or E human nNOS usingessentially the same conditions as above. Proteins may be resolved bySDS-PAGE and ³²P incorporation and the amount of protein determined byCoomassie staining as above.

[0082] The above screening assays which assay the Akt dependentphosphorylation or activation of NOS, preferably eNOS or nNOS, may beused to screen for a wide-variety of agents. For instance, agents whichinhibit the dephosphorylation of NOS (phosphatase inhibitors) at anamino acid corresponding to serine 1179 in bovine eNOS, residue 1177 inhuman eNOS, residue 1412 in rat nNOS, or 1415 in human nNOS may beuseful therapeutic molecules. Similarly, agents which activate Akt orwhich mimic the Akt phosphorylation site on eNOS may be usefultherapeutic molecules.

[0083] Agents that are assayed in the above methods can be randomlyselected or rationally selected or designed. As used herein, an agent issaid to be randomly selected when the agent is chosen randomly withoutconsidering the specific sequences involved in the association of the aprotein of the invention alone or with its associated substrates,binding partners, etc. An example of randomly selected agents is the usea chemical library or a peptide combinatorial library, or a growth brothof an organism.

[0084] As used herein, an agent is said to be rationally selected ordesigned when the agent is chosen on a nonrandom basis which takes intoaccount the sequence of the target site and/or its conformation inconnection with the agent's action. For example, a rationally selectedpeptide agent can be a peptide whose amino acid sequence is similar tothe Akt phosphorylation site in NOS, particularly, peptides or smallmolecules that mimic the NOS phosphorylation state.

[0085] The agents of the present invention can be, as examples,peptides, small molecules, vitamin derivatives, as well ascarbohydrates. A skilled artisan can readily recognize that there is nolimit as to the structural nature of the agents of the presentinvention.

[0086] The peptide agents of the invention can be prepared usingstandard solid phase (or solution phase) peptide synthesis methods, asis known in the art. In addition, the DNA encoding these peptides may besynthesized using commercially available oligonucleotide synthesisinstrumentation and produced recombinantly using standard recombinantproduction systems. The production using solid phase peptide synthesisis necessitated if non-gene-encoded amino acids are to be included.

[0087] Another class of agents of the present invention are antibodiesimmunoreactive with critical positions of proteins of the invention.Antibody agents are obtained by immunization of suitable mammaliansubjects with peptides, containing as antigenic regions, those portionsof the protein intended to be targeted by the antibodies.

[0088] Use of Agents Identified as Modulating eNOS Activity

[0089] The agents of the present invention, such as agents that inhibitthe dephosphorylation of NOS (phosphatase inhibitors) at an amino acidcorresponding to serine 1179 in bovine eNOS, residue 1177 in human eNOS,residue 1412 in rat nNOS, or residue 1415 in human nNOS, as well asagents which activate Akt or which mimic the Akt phosphorylation site onNOS, can be administered via parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, or buccal routes.Alternatively, or concurrently, administration may be by the oral route.The dosage administered will be dependent upon the age, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired. As described below,there are many methods that can readily be adapted to administer suchagents.

[0090] The present invention further provides compositions containingone or more agents of the invention. While individual needs vary, adetermination of optimal ranges of effective amounts of each componentis within the skill of the art. Typical dosages comprise 0.1 to 100mg/kg body wt. The preferred dosages comprise 0.1 to 10 mg/kg body wt.The most preferred dosages comprise 0.1 to 1 mg/kg body wt.

[0091] In addition to the pharmacologically active agent, thecompositions of the present invention may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically for delivery to the siteof action. Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form, forexample, water-soluble salts. In addition, suspensions of the activecompounds as appropriate oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension include, forexample, sodium carboxymethyl cellulose, sorbitol and/or dextran.Optionally, the suspension may also contain stabilizers. Liposomes canalso be used to encapsulate the agent for delivery into the cell.

[0092] The pharmaceutical formulation for systemic administrationaccording to the invention may be formulated for enteral, parenteral ortopical administration. Indeed, all three types of formulations may beused simultaneously to achieve systemic administration of the activeingredient.

[0093] Suitable formulations for oral administration include hard orsoft gelatin capsules, pills, tablets, including coated tablets,elixirs, suspensions, syrups or inhalations and controlled release formsthereof.

[0094] In practicing the methods of this invention, the compounds ofthis invention may be used alone or in combination, or in combinationwith other therapeutic or diagnostic agents. In certain preferredembodiments, the compounds of this invention may be coadministered alongwith other compounds typically prescribed for these conditions accordingto generally accepted medical practice, such as anticoagulant agents,thrombolytic agents, or other antithrombotics, including plateletaggregation inhibitors, tissue plasminogen activators, urokinase,prourokinase, streptokinase, heparin, aspirin, or warfarin. Thecompounds of this invention can be utilized in vivo, ordinarily inmammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, ratsand mice, or in vitro.

[0095] The following working examples specifically point out preferredembodiments of the present invention, and are not to be construed aslimiting in any way the remainder of the disclosure. Other genericconfigurations will be apparent to one skilled in the art.

EXAMPLES

[0096] The following procedures were employed in Examples 1-2

[0097] Cell Transfections: The bovine eNOS, human iNOS, rat nNOS cDNAsin pcDNA3 and HA-tagged wild-type Akt, Akt (K179M) or myr-Akt in pCMV6were generated by standard cloning methods. The myr-nNOS in pcDNA3 wasgenerated by PCR, incorporating a new amino terminus containing the eNOSN-myristoylation consensus site (MGNLKSVG, SEQ ID NO:1) fused in frameto the second amino acid of the nNOS coding sequence. In preliminaryexperiments in COS cells, this construct was N-myristoylated based onincorporation of ³H-myristic acid whereas native nNOS is not andresulted in approximately 60% of the total protein targeted into themembrane fraction of cells whereas only 5-10% of nNOS was membraneassociated in COS cells. Mutation of the putative Akt phosphorylationsites in eNOS were generated using the Quick Change site-directedmutagenesis kit (Stratagene) according to the manufacturersinstructions. All mutants were verified by DNA sequencing. COS-7 cellswere plated (100 mm dish) and transfected with the NOS (7.5-30 mg) andAkt (1 mg) plasmids using calcium phosphate. To balance alltransfections, the expression vector for β-galactosidase cDNA was used.Twenty-four to forty-eight hours after transfection, the expression ofappropriate proteins (40-80 mg) were confirmed by Western blot analysisusing eNOS mAb (9D10, Zymed), HA mAb (12CA5, Boehringer Mannheim), iNOSpAb (Zymed Laboratories) or nNOS mAb (Zymed Laboratories).

[0098] NO release from transfected COS cells: 24-48 hrs aftertransfection, media was processed for the measurement of nitrite (NO₂⁻), the stable breakdown product of NO in aqueous solution, by NOspecific chemiluminescence as described (Sessa et al., 1995). Media wasdeproteinized and samples containing NO₂ ⁻ were refluxed in glacialacetic acid containing sodium iodide. Under these conditions, NO₂ ⁻ wasquantitatively reduced to NO which was quantified by a chemiluminescencedetector after reaction with ozone in a NO analyzer (Sievers, Boulders,Colo.). In all experiments, NO₂ ⁻ release was inhibitable by a NOSinhibitor. In addition, NO₂ ⁻-release from cells transfected with theβ-galactosidase cDNA was subtracted to control for background levels ofNO₂ ⁻ found in serum or media. In some experiments, cGMP accumulation inCOS was used as a bioassay for the production of NO as described.

[0099] NOS activity assays: The conversion of ³H -L-arginine to³H-L-citrulline was used to determine NOS activity in COS cell orendothelial cell lysates as previously described by Garcia-Cardena etal. (1998).

[0100] Phosphorylation studies in vivo and in vitro: For in vivophosphorylation studies, COS cells were transfected with the cDNAs forwild-type or S635, 1179A eNOS and HA-Akt overnight. 36 hrs aftertransfection, cells were placed into dialyzed serum replete,phosphate-free Dulbecco's minimum essential medium supplemented with 80μCi/ml of ³²P orthophosphoric acid for 3 hr. Some cells were pretreatedwith wortmannin (500 nM) in the phosphate-free media for 1 hr and duringthe labeling. Lysates were harvested, eNOS solubilized and partiallypurified by ADP sepharose affinity chromatography as previouslydescribed and the ³²P incorporation into eNOS visualized after SDS-PAGE(7.5%) by autoradiography and the amount of eNOS protein verified byWestern blotting for eNOS. For in vitro phosphorylation studies,recombinant eNOS purified from E. coli was incubated with wild-type orkinase inactive Akt immunoprecipitated from transfected COS cells. eNOSwas incubated with ³²P g-ATP (2 ml, specific activity 3000 Ci/mmol), ATP(50 mM), DTT (1 mM), in a buffer containing HEPES (20 mM, pH=7.4), MnCl₂(10 mM), MgCl₂ (10 mM) and immunoprecipitated Akt for 20 min at roomtemperature.

[0101] In experiments examining the in vitro phosphorylation ofwild-type and mutant eNOS, recombinant Akt (1 mg) purified frombaculovirus infected SF9 cells, was incubated with wild-type or S1179AeNOS (2.4 mg, purified from E. coli) using essentially the sameconditions as above. Proteins were resolved by SDS-PAGE and ³²Pincorporation and the amount of protein determined by Coomassie stainingas above.

[0102] In studies identifying the labeled eNOS peptide,immunoprecipitated Akt was incubated with recombinant eNOS as above. Thesample was run on SDS-PAGE, and the eNOS band digested in gel, and theresultant tryptic fragments purified by RP-HPLC. Peptide mass and ³²Pincorporation were monitored and the prominent labeled peak furtheranalyzed by mass spectrometry. In other experiments, peptidescorresponding to the potential Akt phosphorylation site weresynthesized, purified by HPLC and verified by mass spectrometry (W. M.Keck Biotechnology Resource Center, Yale University School of Medicine).The wild-type peptide was 1174RIRTQSFSLQERHLRGAVPWA1194 (SEQ ID NO:2)and the mutant peptide was identical except S1179 was changed to analanine. In vitro kinase reactions were essentially as described aboveincubating peptides (25 mg) with recombinant Akt (1 mg). Reactions werethen spotted onto phosphocellulose filters and the amount of phosphateincorporated measured by Cerenkov counting.

[0103] Adenoviral infections and NO release in endothelial cells: Bovinelung microvascular endothelial cells (BLMVEC) were cultured in either in100 mm dishes (for basal NO release and NOS activity assays) or C6 wellplates (for stimulated NO) as previously described (Garcia-Cardena etal., 1996a). BLMVEC were infected with 200 MOI of adenovirus containingthe β-galactosidase 29, HA-tagged, inactive phosphorylation mutant Akt(AA-Akt; Alessi et al., 1996) or carboxyl terminal HA-taggedconstitutively active Akt (myr-Akt) for 4 hrs. The virus was removed andcells left to recover for 18 hrs in complete medium. In preliminaryexperiments with the β-galactosidase virus, these conditions wereoptimal for infecting 100% of the cultures. For measurement of basal NOproduction, media was collected for NO release 24 hrs after the initialinfection with virus. For measurement of stimulated NO release, cellswere then washed with serum-free medium followed by stimulation withVEGF (40 ng/ml) for 30 min. In some experiments the calcium dependencyof NOS was determined 24 hrs after adenoviral infection. Infected cellswere lysed in NOS assay buffer containing 1% NP40, and detergent solublematerial used for activity. Lysates were incubated with EGTA bufferedcalcium to yield appropriate amounts of free calcium in the incubation.

[0104] Statistics: Data are expressed as mean±SEM. Comparison's weremade using a two-tailed, Student's t-test or ANOVA with a post-hoc testwhere appropriate. Differences were considered to be significant atp<0.05.

Example 1 Akt Modulates NO Production from eNOS

[0105] To explore the possibility that a downstream effector of PI-3kinase, Akt, could directly influence the production of NO, COS-7 cells(which do not express NOS) were co-transfected with eNOS and wild typeAkt (HA-Akt), or kinase inactive Akt (HA-Akt K179M) and the accumulationof nitrite (NO₂ ⁻) measured by NO specific chemiluminescence.Transfection of eNOS results in an increase in NO₂ ⁻ accumulation, aneffect that is markedly enhanced by co-transfection of wild-type Akt,but not the kinase inactive variant (FIG. 1A). Identical results wereobtained using cGMP as a bioassay for biologically active NO. Underthese experimental conditions, Akt was catalytically active asdetermined by Western blotting with a phospho-Akt specific Ab (whichrecognizes serine 473; not shown) and Akt activity assays (see FIG. 2A).Transfection of a constitutively active form of Akt (myr-Akt) increasescGMP accumulation (assayed in COS cells) from 5.5±0.8 to 11.6±0.9 pmolcGMP/mg protein (in cells transfected with eNOS alone or eNOS withmyr-Akt, respectively) whereas the kinase inactive Akt did not influencecGMP accumulation (5.8±0.8 pmol cGMP, n=4 experiments). As seen in theinset, equal levels of eNOS and Akt were expressed in COS cell lysatessuggesting that Akt modulates eNOS thereby increasing NO productionunder basal conditions.

[0106] eNOS is a dually acylated peripheral membrane protein thattargets into the Golgi region and plasma membrane of endothelial cells(Liu et al., 1997; Garcia-Cardena et al., 1996a; Shaal et al., 1996) andcompartmentalization is required for efficient production of NO inresponse to agonist challenge (Sessa et al., 1995; Liu et al., 1996;Kantor et al., 1996). To examine if eNOS activation by Akt requiresmembrane compartmentalization, COS-7 cells were co-transfected withcDNAs for Akt and a myristoylation, palmitoylation defective mutant ofeNOS (G2A eNOS) and the release of NO quantified. As seen in FIG. 1B,Akt did not activate the non-acylated form of eNOS suggesting thatcompartmentalization of both proteins to the membrane is required fortheir functional interaction. (Downward, et al., 1998). Next, it wasdetermined if Akt could activate structurally similar but distinctsoluble NOS isoforms, neuronal and inducible NOS (nNOS and iNOS,respectively). Co-transfection Akt with nNOS and iNOS did not result ina further increase in NO release demonstrating the specificity of Aktfor eNOS. However, the addition of an N-myristoylation site to nNOS, inorder to enhance its interactions with biological membranes, results inAkt stimulation of nNOS in a manner analogous to that seen with eNOS,suggesting that both isoforms may be susceptible to activation by Aktkinase when membrane anchored.

Example 2 Production of eNOS Mutations

[0107] The above experiments imply that Akt, perhaps via phosphorylationof eNOS, can modulate NO release from intact cells. Indeed, two putativeAkt phosphorylation motifs (RXRXXS/T) are present in eNOS (serines 635and 1179 in bovine eNOS or serines 633 and 1177 in human eNOS) and onemotif present in nNOS (serines 1412 in rat and 1415 human nNOS), with noobvious motifs found in iNOS. To examine if eNOS is a potentialsubstrate for Akt phosphorylation in vitro, COS cells were transfectedwith HA-Akt or HA-Akt (K179M) and kinase activity assessed usingrecombinant eNOS as a substrate. As seen in FIG. 2A, the active kinasephosphorylates histone 2B and eNOS (69.3±2.9 and 115.4±3.8 pmol ofATP/nmol substrate, respectively, n=3), whereas the inactive Akt did notsignificantly increase histone or eNOS phosphorylation. To elucidate ifthe putative Akt phosphorylation sites in eNOS were responsible for theincorporation of ³²P, the two serines were mutated to alanine residuesand the ability of Akt to stimulate wild-type and mutant eNOSphosphorylation examined in intact COS cells. Transfected cells werelabeled with ³²P -orthophosphate, eNOS partially purified byADP-sepharose affinity chromatography, and the phosphorylation state andprotein levels quantified. As seen in FIG. 2B, co-expression of Aktresults in a 2 fold enhancement in the phosphorylation of eNOS relativeto non-stimulated cells. Pretreatment of eNOS/Akt transfected cells withwortmannin abolished the Akt induced increase in phosphorylation.Moreover, mutation of serine 635 and 1179 to alanine residues abolishedAkt dependent phosphorylation of eNOS suggesting that these residuescould serve as potential phosphorylation sites in intact cells.

[0108] To directly identify the residues phosphorylated by Akt,wild-type eNOS was incubated with immunopurified Akt and the sites ofphosphorylation determined by HPLC followed by MALDi-mass spectrometry(MALDi-MS). As seen in FIG. 2C, the primary ³²P-labeled trypticphosphopeptide co-elutes with a synthetic phosphopeptide (amino acids1177-1185 with phosphoserine at position 1179) and has the identicalmass ion as determined by linear mode MS. Using reflectron mode MALDi-MSmonitoring, both the labeled tryptic peptide and the standardphosphopeptide demonstrated a loss of HP₃O₄ indicating that the trypticpeptide was phosphorylated. In addition, mutation of S1179 to A markedlyreduces Akt-dependent phosphorylation of eNOS compared to the wild-typeprotein (FIG. 2D). Identical results were obtained utilizing peptides(amino acids 1174-1194) derived from wild-type or eNOS S1179A assubstrates for recombinant Akt (wild-type peptide incorporated 24.6±3.7nmol phosphate/mg compared to the alanine mutant peptide whichincorporated 0.22±0.02 nmol phosphate/mg, n=5). Collectively, these datademonstrate that eNOS is a substrate for Akt and that the primary siteof phosphorylation is serine 1179 (serine 1177 in human eNOS).

[0109] Next we examined the functional significance of the putative Aktphosphorylation site at serine 635 and the identified site at serine1179. Transfection of COS cells with the double mutant eNOS S635/1179Aabolishes Akt dependent NO release. Mutation of serine 635 to alaninedid not attenuate NO release whereas eNOS S1179A abolishes Akt dependentactivation of eNOS (FIG. 3). These results suggest that serine 1179 isfunctionally important for NO release. Mutation of serine 1179 intoaspartic acid (eNOS S1179D) to substitute for the negative chargeafforded by the addition of phosphate, partially mimics the activationstate induced by Akt (S1177D in human eNOS). All site directed mutantswere amply expressed (see inset Western blots) and retained NOScatalytic activity in cell lysates (in COS cells transfected with eNOSonly, NOS activity was 85.3±27.0, 71.9±2.9, 80.8±23.2 and 131.8±36.7pmol L-citrulline generated/mg protein from lysates of COS cellsexpressing wild type, S1179A, S635, 1179A and eNOS S1179D, respectively,n=3 experiments).

[0110] To examine if Akt mediates NO release from endothelial cells,bovine lung microvascular endothelial cells (BLMVEC) were infected withadenoviruses expressing activated Akt (myr-Akt), activation deficientAkt (AA-Akt) or β-galactosidase as a control and the accumulation of NOmeasured. As seen in FIG. 4A, myr-Akt stimulates the basal production ofNO from BLMVEC, whereas cells infected with β-galactosidase oractivation deficient Akt released low levels of NO that were close tothe limits of detection. These data in conjunction with similar resultsin COS cells suggests that Akt phosphorylation of eNOS is sufficient toregulate NO production at resting levels of calcium. Indeed, NOSactivity measured in lysates from myr-Akt infected BLMVEC demonstratesthat the sensitivity of the enzyme to activation by calcium, assayed ata fixed calmodulin concentration, is enhanced relative to NOS activityseen in BLMVEC infected with the β-galactosidase virus (FIG. 4B).Interestingly, the calcium sensitivity of NOS activity in cells infectedwith activation deficient Akt was greatly suppressed relative to bothmyr-Akt and β-galactosidase infected cells.

[0111] Treatment of endothelial cells with VEGF is known to activate Akt23 and the release of NO through a mechanism partially blocked byinhibitors of PI-3 kinase (Papapetropoulos et al., 1997). To examine thefunctional link between VEGF as an agonist for NO release and Aktactivation BLMVEC were infected with adenoviruses for myr-Akt, AA-Akt orβ-galactosidase and VEGF stimulated NO release quantified. As seen inFIG. 4C, infection of endothelial cells with myr-Akt enhances VEGFdriven NO production while the AA-Akt attenuates NO release. Theseresults imply that Akt participates in the signal transduction eventsrequired for both basal and stimulated NO production in endothelialcells.

[0112] Collectively these data demonstrate that Akt can phosphorylateeNOS on serine 1179 (serine 1177 in human eNOS) and that phosphorylationenhances the ability of the enzyme to generate NO.

Example 3 Materials and Methods

[0113] eNOS Constructs and Protein Purification—Wild type bovine eNOS inthe plasmid pCW was expressed as described previously with groELS in E.coli BL21 cells (Martasek et al., 1996). The S1179D mutant eNOS forexpression in E. coli was generated as follows. eNOS S1179D in pcDNA 3(Fulton et al., 1999) was digested with XhoI/XbaI, subcloned into theidentical sites of eNOS in pCW, and co-expressed with groELS. Isolationof recombinant eNOS was preformed as reported previously (Roman et al.,1995; Martasek et al., 1999), with the following modifications. eNOS waseluted from 2′5′-ADP Sepharose with either 10 mM NADPH or 10 mM 2′-AMP.The amount of eNOS was quantitated using the peak absorbance at 409-412nm, with an extinction coefficient for heme content of 0.1 μM⁻¹ cm⁻¹.The purity of eNOS was determined by 7.5% SDS-PAGE followed by Coomassiestaining. Low temperature SDS-PAGE was performed identically, exceptthat samples were not boiled and the electrophoresis was carried out at4° C. in a slurry of ice/water (Klatt et al., 1995). In experiments inwhich NOS cofactors (L-arginine, calmodulin, and NADPH) were titrated,they were omitted from the purification and storage of the enzymes andwere incubated as described below.

[0114] Assay for NOS Activity—NO production was measured by thehemoglobin capture assay as described (Kelm et al., 1988). Briefly, thereaction mixture contained eNOS (0.5-2.5 μg), oxyhemoglobin (8 μM),L-arginine (100 μM), BH4 (5 μM), CaCl2 (120 μM), calmodulin (120-200nM), and NADPH (100 μM) in HEPES buffer (50 mM), pH 7.4. In thedetermination of calcium EC50 value for eNOS, the above reaction mixturewas modified as follows: MOPS buffer (10 mM, pH 7.6), KCl (100 mM) andCaM (250 nM) were substituted. Under these conditions, free calcium wascalculated using the WinMAXC program, version 1.8 (Stanford University),with a Kd of 2.2×10⁻⁸ M. The precise free calcium concentration wasachieved by mixing an appropriate proportion of 10 mM K₂EGTA and 10 mMCaEGTA stock solutions (Molecular Probes). NOS activity was monitoredfor linearity over 2 min at 401 nm, and NO production was calculatedbased on the change of absorbance using the extinction coefficient of 60mM⁻¹ cm⁻¹. All reactions were carried out at 23° C., and each data pointrepresents 3-8 observations. The extinction coefficient of 0.0033 μM⁻¹cm⁻¹ at 276 nm was used for determination of calmodulin concentration.The production of NO using this method was completely blocked by theaddition of nitro-L-arginine (1 mM). When the inactivation of eNOS wasdetermined by the addition of EGTA (200-800 μM) to the reaction mixture,chelator was added 1 min after initiation of the reaction by NADPH.Identical conditions were used when NADPH-cytochrome c reductaseactivity was examined. These reactions contained CaM (120 nM) and CaCl₂(200 μM) in a 0.5-ml volume with eNOS (0.5 μg).

[0115] The conversion of L-arginine to L-citrulline was assayed asdescribed previously by Bredt et al. (1990). Briefly, eNOS (0.25-2 μg)was incubated for 3-10 min at 23° C. in the following reaction mixture:3 pmol of L-[3H ]arginine (55 Ci/mmol), 10-300 μM arginine, 1 mM NADPH,120-200 nM calmodulin, 2 mM CaCl2, and 30 μM BH4 in a final reactionvolume of 50-100 μl. The reaction was terminated by the addition of 0.5ml of 20 mM HEPES, pH 5.5, containing 2 mM EGTA and EDTA. The reactionmixture was placed over Dowex AG50WX8, and the flow-through was countedon a Packard 1500 liquid scintillation analyzer.

[0116] Assays for Reductase Activity—NADPH-cytochrome c reductaseactivity and 2,6-dichlorophenolindophenol (DCIP) reduction were measuredas a change in absorbance at 550nm as described previously by Martaseket al. (1999) and Masters et al. (1967) using an extinction coefficientof 0.021 μM⁻¹ cm⁻¹ for both cytochrome c and DCIP. Briefly, a reactionmixture (1 ml) contained either cytochrome c (90 μM); DCIP (36 μM),HEPES buffer (50 mM) at pH 7.6, NaCl (250 mM), NADPH (100 μM),calmodulin (120 nM), and CaCl2 (200 μM); or other substances asindicated. The reaction was monitored for 60 s (at 23° C.) after theaddition of eNOS. When inactivation of reductase activity was determinedby the addition of EGTA (200-800 μM), chelator was added 1 min afterinitiation of the reaction and monitored for an additional 1 min. Thereaction contained HEPES buffer (50 mM) at pH 7.6, CaM (120 nM), andCaCl2 (200 μM) and was initiated with NADPH (100 μM. No NaCl was addedin experiments that examined EGTA inactivation of eNOS to mimic theconditions used in the hemoglobin capture experiments. The addition ofNOS inhibitors did not influence the rate of cytochrome c reduction (notshown). Determination of the calcium EC50 for eNOS was performed asdescribed above for the hemoglobin capture assay.

[0117] Data Analysis and Statistics—All data were expressed as mean±S.E.At least triplicate determinations were performed with a minimum ofthree different batches of enzymes for each data set. Wild type andmutant enzymes were purified simultaneously to control for activityvariations between preparations. Statistical significance was determinedusing Student's t test, and p<0.05 was considered statisticallysignificant.

Results

[0118] Expression and Purification of eNOS—Both wild type and S1179DeNOS were expressed and purified from E. coli. In a culture of 1.6liters, approximately 2.5-4.0 mg of eNOS was typically recovered using2′5′-ADP Sepharose 4B chromatography. As seen in FIG. 5A, both enzymeswere >90% pure based on Coomassie staining. These results are typical,as seen from seven independent preparations of both wild type and S1179DeNOS prepared side-by-side. Both enzymes were primarily in their dimericform, as shown by low temperature SDS-PAGE (FIG. 5B).

[0119] eNOS S1179D Has Greater NO Synthase and Reductase Activities ThanDoes Wild Type eNOS—Next, the activities of wild type and S1179D eNOSwere compared by measuring the rate of NO production. S1179D eNOSexhibited a higher turnover number (under optimal conditions) ascompared with wild type enzyme (84±6 versus 27±1 min⁻¹, n=6 separate andpaired preparations of enzymes). The Km values with L-arginine weresimilar for wild type and S1179D eNOS (FIG. 6A, 1.8 versus 2.5 μM,respectively; see Table I). TABLE I Kinetic parameters for wild type andS1179D eNOS Catalytic Substrate/cofactors Assay determination Wild typeS1179D Arginine Hemoglobin K_(cat)  27 ± 1 min⁻¹  84 ± 6 min⁻¹ captureK_(m)  1.8 μM  2.5 μM Hemoglobin K_(cat)  17 ± 1 min⁻¹  53 ± 3 min⁻¹capture K_(m)   8 μM   36 μM Cytochrome c K_(cat) + CaM  460 ± 18 min⁻¹ 840 ± 59 min⁻¹ K_(cat) − CaM  70 ± 5 min⁻¹  290 ± 9 min⁻¹ K_(m) + CaM0.75 μM  1.9 μM NADPH −CaM 0.40 μM  2.0 μM L-Citrulline K_(cat)  22 ± 2min⁻¹  43 ± 2 min⁻¹ EC₅₀   8 nM   7 nM Cytochrome c K_(cat)  620 ± 78min⁻¹ 1140 ± 75 min⁻¹ CaM EC₅₀   13 nM   21 mM Hemoglobin K_(cat)  58 ±1 min⁻¹  100 ± 3 min⁻¹ capture (100 mM EC₅₀  310 nM  250 nM KCl)Cytochrome c K_(cat) 1909 ± 33 min⁻¹ 3798 ± 54 min⁻¹ Ca2+ (100 mM EC₅₀ 290 nM  220 nM KCl)

[0120] Because the rate of electron flux from the reductase domain tothe oxygenase domain is critical for NOS catalysis, the increase inS1179D eNOS activity was examined to determine whether it could beattributed to enhanced reductase activity. When both DCIP and cytochromec reduction were examined, a significant increase in activity for S1179Dcompared with wild type eNOS was observed (FIG. 6B). Furthermore, thisincrease was accentuated by the presence of CaM, which increased theoverall activity for both enzymes. Basal cytochrome c reduction, in theabsence of CaM, was 4-fold higher for S1179D compared with wild typeeNOS. The magnitude of CaM-stimulated cytochrome c reduction was higherfor S1179D eNOS (749±35 versus 1272±55 min⁻¹ for wild type and S1179DeNOS, respectively, n=3-5); however, the level of stimulation by CaM was8-fold for wild type eNOS compared with only 3-fold for S1179D eNOS.

[0121] Next, it was determined whether S1179D eNOS produces moresuperoxide than wild type eNOS, which could reduce cytochrome c. Asexpected, no superoxide dismutase inhibitable cytochrome c reduction wasobserved (as an index of superoxide anion generation) in the absence ofCaM, as reported earlier for wild type eNOS (86±6 versus 95±8 min⁻¹) andfor S1179D eNOS (278±9 versus 288±7 min⁻¹, n=3-5). However, in thepresence of CaM, superoxide dismutase reduced the rate of cytochrome creduction for both wild type (610±51 versus 866±8 min⁻¹) and S1179D(1179±43 versus 1518±19 min⁻¹) eNOS. The relative decrease in cytochromec activity in the presence of superoxide dismutase was similar with bothenzymes (30% for wild type and 22% for S1179D eNOS), suggesting that theenhanced reductase activity of S1179D compared with wild type eNOS(assayed by cytochrome c reduction) was not due to uncoupling of theenzyme.

[0122] NADPH-dependent NO Formation and Reductase Activities Are NotDifferent for Wild Type and S1179D eNOS—The NADPH dependence of NOproduction and cytochrome c reduction were examined because the NADPHbinding site lies close in proximity to the Ser-1179 in eNOS. S1179DeNOS had a higher maximum turnover number (k_(cat)) than did wild typeenzyme based on NO production, assayed in the presence of CaM (FIG. 7A).The increased k_(cat) was associated with a 4-fold increase in the Kmfor NADPH for S1179D eNOS compared with wild type eNOS (36 versus 8 μM,respectively). The k_(cat) for NADPH-dependent cytochrome c reduction inthe absence of CaM was greater for eNOS S1179D than wild type eNOS (290versus 70 min⁻¹, respectively; FIG. 7B). In the presence of CaM, thek_(cat) for cytochrome c reduction was considerably higher for S1179Dcompared with wild type eNOS (840 versus 460 min⁻¹, respectively). TheKm values for NADPH were unchanged in the absence or presence of CaM(0.40 and 0.75 μM for wild type eNOS and 2.0 and 1.9 μM for S1179D eNOSin the absence and presence of CaM, respectively).

[0123] EC50 Values for CaM Are Unchanged between Wild Type and S1179DeNOS—To assess whether the increased activity of S1179D eNOS wasattributable to changes in the affinity of the enzyme for CaM, thekinetics of NOS activity and cytochrome c reduction assayed in thepresence of all NOS cofactors in excess as a function of CaMconcentration were examined. The kcat for CaM activation of NOS activitywas 22 min⁻¹ for wild type and 43 min⁻¹ for S1179D eNOS. Transformationof the data, normalizing for the differences in k_(cat), revealed aslight shift in the curve to the left for S1179D eNOS but littledifference in the EC50 values for CaM, consistent with reported data onphospho-eNOS (Mitchell et al., 1999). The EC50 values were 8 nM for wildtype and 7 nM S1179D eNOS (FIG. 8A). NADPH-mediated cytochrome creduction was measured. The k_(cat) for CaM activation of cytochrome creduction was about 2-fold higher for S1179D eNOS compared with wildtype enzyme (1140 and 620 min⁻¹ for S1179D and wild type eNOS,respectively). Transformation of the data normalized for the differencesin k_(cat), revealed small differences in the EC50 values for CaMbetween wild type and S1179D eNOS (21 versus 13 nM; FIG. 8B).

[0124] Comparison of Calcium Activation and Inactivation of eNOS—Inprevious experiments, it was demonstrated that the “apparent calciumsensitivity” of eNOS was enhanced in cells expressing either a majorityof phospho-eNOS or S1179D eNOS, suggesting that phosphorylation changedthe affinity of calcium/CaM activation (Dimmeler et al., 1999; Fulton etal. 1999). As seen in FIG. 8C, after normalization for the differencesin maximal activity, the calcium dependence was slightly increased forS1179D eNOS (p<0.05, two-way analysis of variance). The EC50 values forcalcium with wild type and S1179D eNOS were slightly different also (310and 250 nM calcium, respectively), as determined by NO production (inthe presence of 250 nM CaM). As seen in the inset to FIG. 8C, increasingconcentrations of free calcium did indeed enhance S1179D eNOS turnoverto a greater extent then that seen with wild type enzyme. Furthermore,the EC50 value for calcium assaying cytochrome c reduction were similarto those obtained measuring NO production (FIG. 8D; 290 and 220 nM forwild type and S1179D eNOS, respectively). Again, the Vmax for calciumactivation of S1179D eNOS turnover was greater than wild type (FIG. 4D,inset).

[0125] To examine whether the inactivation of S1179D eNOS was differentthan that of wild type enzyme, the decay in eNOS activity afterchelation of calcium with EGTA was measured. In these experiments, allNOS cofactors were added in the presence of calcium (200 μM), and NOproduction was monitored for 1 min, followed by the addition ofdifferent concentrations of EGTA and monitoring for an additional 1 min.As seen in FIG. 9A, the addition of EGTA to wild type and S1179D eNOSreduced NO production in a concentration-dependent manner. However, NOproduction from S1179D eNOS was less sensitive to the addition of EGTA;i.e. wild type eNOS activity declined more rapidly at lowerconcentrations of EGTA than did S1179D eNOS activity. The greatestdifference in activity between the enzymes was seen at 400 μM EGTA.Furthermore, at 600 μM EGTA, no activity was detected for wild typeeNOS, whereas residual activity was still detected for S1179D eNOS.Similar results were obtained using cytochrome c reduction (FIG. 9B),with significant differences in activity seen with 400 and 600 μMchelator added to the reaction. However, at the highest concentration ofEGTA, residual reductase activity was found for both wild type andS1179D eNOS.

[0126] In summary, bovine endothelial nitric oxide synthase (eNOS) isphosphorylated directly by the protein kinase Akt at serine 1179 (Fultonet al., 1999) and human endothelial nitric oxide synthease isphosphorylated directly by the protein kinase Akt at serine 1177(Dimmeler et al., 1999). Mutation of residue 1179 in bovine eNOS to thenegatively charged aspartate increases nitric oxide (NO) productionconstitutively, in the absence of agonist challenge.

[0127] It should be understood that the foregoing discussion andexamples merely present a detailed description of certain preferredembodiments. It therefore should be apparent to those of ordinary skillin the art that various modifications and equivalents can be madewithout departing from the spirit and scope of the invention. Allreferences, articles and patents identified above or below are hereinincorporated by reference in their entirety.

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1 9 1 8 PRT Bos sp. 1 Met Gly Asn Leu Lys Ser Val Gly 1 5 2 21 PRT Bossp. 2 Arg Ile Arg Thr Gln Ser Phe Ser Leu Gln Glu Arg His Leu Arg Gly 15 10 15 Ala Val Pro Trp Ala 20 3 1203 PRT Homo sapiens 3 Met Gly Asn LeuLys Ser Val Ala Gln Glu Pro Gly Pro Pro Cys Gly 1 5 10 15 Leu Gly LeuGly Leu Gly Leu Gly Leu Cys Gly Lys Gln Gly Pro Ala 20 25 30 Thr Pro AlaPro Glu Pro Ser Arg Ala Pro Ala Ser Leu Leu Pro Pro 35 40 45 Ala Pro GluHis Ser Pro Pro Ser Ser Pro Leu Thr Gln Pro Pro Glu 50 55 60 Gly Pro LysPhe Pro Arg Val Lys Asn Trp Glu Val Gly Ser Ile Thr 65 70 75 80 Tyr AspThr Leu Ser Ala Gln Ala Gln Gln Asp Gly Pro Cys Thr Pro 85 90 95 Arg ArgCys Leu Gly Ser Leu Val Phe Pro Arg Lys Leu Gln Gly Arg 100 105 110 ProSer Pro Gly Pro Pro Ala Pro Glu Gln Leu Leu Ser Gln Ala Arg 115 120 125Asp Phe Ile Asn Gln Tyr Tyr Ser Ser Ile Lys Arg Ser Gly Ser Gln 130 135140 Ala His Glu Gln Arg Leu Gln Glu Val Glu Ala Glu Val Ala Ala Thr 145150 155 160 Gly Thr Tyr Gln Leu Arg Glu Ser Glu Leu Val Phe Gly Ala LysGln 165 170 175 Ala Trp Arg Asn Ala Pro Arg Cys Val Gly Arg Ile Gln TrpGly Lys 180 185 190 Leu Gln Val Phe Asp Ala Arg Asp Cys Arg Ser Ala GlnGlu Met Phe 195 200 205 Thr Tyr Ile Cys Asn His Ile Lys Tyr Ala Thr AsnArg Gly Asn Leu 210 215 220 Arg Ser Ala Ile Thr Val Phe Pro Gln Arg CysPro Gly Arg Gly Asp 225 230 235 240 Phe Arg Ile Trp Asn Ser Gln Leu ValArg Tyr Ala Gly Tyr Arg Gln 245 250 255 Gln Asp Gly Ser Val Arg Gly AspPro Ala Asn Val Glu Ile Thr Glu 260 265 270 Leu Cys Ile Gln His Gly TrpThr Pro Gly Asn Gly Arg Phe Asp Val 275 280 285 Leu Pro Leu Leu Leu GlnAla Pro Asp Glu Pro Pro Glu Leu Phe Leu 290 295 300 Leu Pro Pro Glu LeuVal Leu Glu Val Pro Leu Glu His Pro Thr Leu 305 310 315 320 Glu Trp PheAla Ala Leu Gly Leu Arg Trp Tyr Ala Leu Pro Ala Val 325 330 335 Ser AsnMet Leu Leu Glu Ile Gly Gly Leu Glu Phe Pro Ala Ala Pro 340 345 350 PheSer Gly Trp Tyr Met Ser Thr Glu Ile Gly Thr Arg Asn Leu Cys 355 360 365Asp Pro His Arg Tyr Asn Ile Leu Glu Asp Val Ala Val Cys Met Asp 370 375380 Leu Asp Thr Arg Thr Thr Ser Ser Leu Trp Lys Asp Lys Ala Ala Val 385390 395 400 Glu Ile Asn Val Ala Val Leu His Ser Tyr Gln Leu Ala Lys ValThr 405 410 415 Ile Val Asp His His Ala Ala Thr Ala Ser Phe Met Lys HisLeu Glu 420 425 430 Asn Glu Gln Lys Ala Arg Gly Gly Cys Pro Ala Asp TrpAla Trp Ile 435 440 445 Val Pro Pro Ile Ser Gly Ser Leu Thr Pro Val PheHis Gln Glu Met 450 455 460 Val Asn Tyr Phe Leu Ser Pro Ala Phe Arg TyrGln Pro Asp Pro Trp 465 470 475 480 Lys Gly Ser Ala Ala Lys Gly Thr GlyIle Thr Arg Lys Lys Thr Phe 485 490 495 Lys Glu Val Ala Asn Ala Val LysIle Ser Ala Ser Leu Met Gly Thr 500 505 510 Val Met Ala Lys Arg Val LysAla Thr Ile Leu Tyr Gly Ser Glu Thr 515 520 525 Gly Arg Ala Gln Ser TyrAla Gln Gln Leu Gly Arg Leu Phe Arg Lys 530 535 540 Ala Phe Asp Pro ArgVal Leu Cys Met Asp Glu Tyr Asp Val Val Ser 545 550 555 560 Leu Glu HisGlu Thr Leu Val Leu Val Val Thr Ser Thr Phe Gly Asn 565 570 575 Gly AspPro Pro Glu Asn Gly Glu Ser Phe Ala Ala Ala Leu Met Glu 580 585 590 MetSer Gly Pro Tyr Asn Ser Ser Pro Arg Pro Glu Gln His Lys Ser 595 600 605Tyr Lys Ile Arg Phe Asn Ser Ile Ser Cys Ser Asp Pro Leu Val Ser 610 615620 Ser Trp Arg Arg Lys Arg Lys Glu Ser Ser Asn Thr Asp Ser Ala Gly 625630 635 640 Ala Leu Gly Thr Leu Arg Phe Cys Val Phe Gly Leu Gly Ser ArgAla 645 650 655 Tyr Pro His Phe Cys Ala Phe Ala Arg Ala Val Asp Thr ArgLeu Glu 660 665 670 Glu Leu Gly Gly Glu Arg Leu Leu Gln Leu Gly Gln GlyAsp Glu Leu 675 680 685 Cys Gly Gln Glu Glu Ala Phe Arg Gly Trp Ala GlnAla Ala Phe Gln 690 695 700 Ala Ala Cys Glu Thr Phe Cys Val Gly Glu AspAla Lys Ala Ala Ala 705 710 715 720 Arg Asp Ile Phe Ser Pro Lys Arg SerTrp Lys Arg Gln Arg Tyr Arg 725 730 735 Leu Ser Ala Gln Ala Glu Gly LeuGln Leu Leu Pro Gly Leu Ile His 740 745 750 Val His Arg Arg Lys Met PheGln Ala Thr Ile Arg Ser Val Glu Asn 755 760 765 Leu Gln Ser Ser Lys SerThr Arg Ala Thr Ile Leu Val Arg Leu Asp 770 775 780 Thr Gly Gly Gln GluGly Leu Gln Tyr Gln Pro Gly Asp His Ile Gly 785 790 795 800 Val Cys ProPro Asn Arg Pro Gly Leu Val Glu Ala Leu Leu Ser Arg 805 810 815 Val GluAsp Pro Pro Ala Pro Thr Glu Pro Val Ala Val Glu Gln Leu 820 825 830 GluLys Gly Ser Pro Gly Gly Pro Pro Pro Gly Trp Val Arg Asp Pro 835 840 845Arg Leu Pro Pro Cys Thr Leu Arg Gln Ala Leu Thr Phe Phe Leu Asp 850 855860 Ile Thr Ser Pro Pro Ser Pro Gln Leu Leu Arg Leu Leu Ser Thr Leu 865870 875 880 Ala Glu Glu Pro Arg Glu Gln Gln Glu Leu Glu Ala Leu Ser GlnAsp 885 890 895 Pro Arg Arg Tyr Glu Glu Trp Lys Trp Phe Arg Cys Pro ThrLeu Leu 900 905 910 Glu Val Leu Glu Gln Phe Pro Ser Val Ala Leu Pro AlaPro Leu Leu 915 920 925 Leu Thr Gln Leu Pro Leu Leu Gln Pro Arg Tyr TyrSer Val Ser Ser 930 935 940 Ala Pro Ser Thr His Pro Gly Glu Ile His LeuThr Val Ala Val Leu 945 950 955 960 Ala Tyr Arg Thr Gln Asp Gly Leu GlyPro Leu His Tyr Gly Val Cys 965 970 975 Ser Thr Trp Leu Ser Gln Leu LysPro Gly Asp Pro Val Pro Cys Phe 980 985 990 Ile Arg Gly Ala Pro Ser PheArg Leu Pro Pro Asp Pro Ser Leu Pro 995 1000 1005 Cys Ile Leu Val GlyPro Gly Thr Gly Ile Ala Pro Phe Arg Gly 1010 1015 1020 Phe Trp Gln GluArg Leu His Asp Ile Glu Ser Lys Gly Leu Gln 1025 1030 1035 Pro Thr ProMet Thr Leu Val Phe Gly Cys Arg Cys Ser Gln Leu 1040 1045 1050 Asp HisLeu Tyr Arg Asp Glu Val Gln Asn Ala Gln Gln Arg Gly 1055 1060 1065 ValPhe Gly Arg Val Leu Thr Ala Phe Ser Arg Glu Pro Asp Asn 1070 1075 1080Pro Lys Thr Tyr Val Gln Asp Ile Leu Arg Thr Glu Leu Ala Ala 1085 10901095 Glu Val His Arg Val Leu Cys Leu Glu Arg Gly His Met Phe Val 11001105 1110 Cys Gly Asp Val Thr Met Ala Thr Asn Val Leu Gln Thr Val Gln1115 1120 1125 Arg Ile Leu Ala Thr Glu Gly Asp Met Glu Leu Asp Glu AlaGly 1130 1135 1140 Asp Val Ile Gly Val Leu Arg Asp Gln Gln Arg Tyr HisGlu Asp 1145 1150 1155 Ile Phe Gly Leu Thr Leu Arg Thr Gln Glu Val ThrSer Arg Ile 1160 1165 1170 Arg Thr Gln Ser Phe Ser Leu Gln Glu Arg GlnLeu Arg Gly Ala 1175 1180 1185 Val Pro Trp Ala Phe Asp Pro Pro Gly SerAsp Thr Asn Ser Pro 1190 1195 1200 4 1205 PRT Bovine 4 Met Gly Asn LeuLys Ser Val Gly Gln Glu Pro Gly Pro Pro Cys Gly 1 5 10 15 Leu Gly LeuGly Leu Gly Leu Gly Leu Cys Gly Lys Gln Gly Pro Ala 20 25 30 Ser Pro AlaPro Glu Pro Ser Arg Ala Pro Ala Pro Ala Thr Pro His 35 40 45 Ala Pro AspHis Ser Pro Ala Pro Asn Ser Pro Thr Leu Thr Arg Pro 50 55 60 Pro Glu GlyPro Lys Phe Pro Arg Val Lys Asn Trp Glu Leu Gly Ser 65 70 75 80 Ile ThrTyr Asp Thr Leu Cys Ala Gln Ser Gln Gln Asp Gly Pro Cys 85 90 95 Thr ProArg Cys Cys Leu Gly Ser Leu Val Leu Pro Arg Lys Leu Gln 100 105 110 ThrArg Pro Ser Pro Gly Pro Pro Pro Ala Glu Gln Leu Leu Ser Gln 115 120 125Ala Arg Asp Phe Ile Asn Gln Tyr Tyr Ser Ser Ile Lys Arg Ser Gly 130 135140 Ser Gln Ala His Glu Glu Arg Leu Gln Glu Val Glu Ala Glu Val Ala 145150 155 160 Ser Thr Gly Thr Tyr His Leu Arg Glu Ser Glu Leu Val Phe GlyAla 165 170 175 Lys Gln Ala Trp Arg Asn Ala Pro Arg Cys Val Gly Arg IleGln Trp 180 185 190 Gly Lys Leu Gln Val Phe Asp Ala Arg Asp Cys Ser SerAla Gln Glu 195 200 205 Met Phe Thr Tyr Ile Cys Asn His Ile Lys Tyr AlaThr Asn Arg Gly 210 215 220 Asn Leu Arg Ser Ala Ile Thr Val Phe Pro GlnArg Ala Pro Gly Arg 225 230 235 240 Gly Asp Phe Arg Ile Trp Asn Ser GlnLeu Val Arg Tyr Ala Gly Tyr 245 250 255 Arg Gln Gln Asp Gly Ser Val ArgGly Asp Pro Ala Asn Val Glu Ile 260 265 270 Thr Glu Leu Cys Ile Gln HisGly Trp Thr Pro Gly Asn Gly Arg Phe 275 280 285 Asp Val Leu Pro Leu LeuLeu Gln Ala Pro Asp Glu Ala Pro Glu Leu 290 295 300 Phe Val Leu Pro ProGlu Leu Val Leu Glu Val Pro Leu Glu His Pro 305 310 315 320 Thr Leu GluTrp Phe Ala Ala Leu Gly Leu Arg Trp Tyr Ala Leu Pro 325 330 335 Ala ValSer Asn Met Leu Leu Glu Ile Gly Gly Leu Glu Phe Ser Ala 340 345 350 AlaPro Phe Ser Gly Trp Tyr Met Ser Thr Glu Ile Gly Thr Arg Asn 355 360 365Leu Cys Asp Pro His Arg Tyr Asn Ile Leu Glu Asp Val Ala Val Cys 370 375380 Met Asp Leu Asp Thr Arg Thr Thr Ser Ser Leu Trp Lys Asp Lys Ala 385390 395 400 Ala Val Glu Ile Asn Leu Ala Val Leu His Ser Phe Gln Leu AlaLys 405 410 415 Val Thr Ile Val Asp His His Ala Ala Thr Val Ser Phe MetLys His 420 425 430 Leu Asp Asn Glu Gln Lys Ala Arg Gly Gly Cys Pro AlaAsp Trp Ala 435 440 445 Trp Ile Val Pro Pro Ile Ser Gly Ser Leu Thr ProVal Phe His Gln 450 455 460 Glu Met Val Asn Tyr Ile Leu Ser Pro Ala PheArg Tyr Gln Pro Asp 465 470 475 480 Pro Trp Lys Gly Ser Ala Thr Lys GlyAla Gly Ile Thr Arg Lys Lys 485 490 495 Thr Phe Lys Glu Val Ala Asn AlaVal Lys Ile Ser Ala Ser Leu Met 500 505 510 Gly Thr Leu Met Ala Lys ArgVal Lys Ala Thr Ile Leu Tyr Ala Ser 515 520 525 Glu Thr Gly Arg Ala GlnSer Tyr Ala Gln Gln Leu Gly Arg Leu Phe 530 535 540 Arg Lys Ala Phe AspPro Arg Val Leu Cys Met Asp Glu Tyr Asp Val 545 550 555 560 Val Ser LeuGlu His Glu Ala Leu Val Leu Val Val Thr Ser Thr Phe 565 570 575 Gly AsnGly Asp Pro Pro Glu Asn Gly Glu Ser Phe Ala Ala Ala Leu 580 585 590 MetGlu Met Ser Gly Pro Tyr Asn Ser Ser Pro Arg Pro Glu Gln His 595 600 605Lys Ser Tyr Lys Ile Arg Phe Asn Ser Val Ser Cys Ser Asp Pro Leu 610 615620 Val Ser Ser Trp Arg Arg Lys Arg Lys Glu Ser Ser Asn Thr Asp Ser 625630 635 640 Ala Gly Ala Leu Gly Thr Leu Arg Phe Cys Gly Phe Gly Leu GlySer 645 650 655 Arg Ala Tyr Pro His Phe Cys Ala Phe Ala Arg Ala Val AspThr Arg 660 665 670 Leu Glu Glu Leu Gly Gly Glu Arg Leu Leu Gln Leu GlyGln Gly Asp 675 680 685 Glu Leu Cys Gly Gln Glu Glu Ala Phe Arg Gly TrpAla Lys Ala Ala 690 695 700 Phe Gln Ala Ser Cys Glu Thr Phe Cys Val GlyGlu Glu Ala Lys Ala 705 710 715 720 Arg Pro Gln Asp Ile Phe Ser Pro LysArg Ser Trp Lys Arg Gln Arg 725 730 735 Tyr Arg Leu Ser Thr Gln Ala GluGly Leu Gln Leu Leu Pro Gly Leu 740 745 750 Ile His Val His Arg Arg LysMet Phe Gln Ala Thr Val Leu Ser Val 755 760 765 Glu Asn Leu Gln Ser SerLys Ser Thr Arg Ala Thr Ile Leu Val Arg 770 775 780 Leu Asp Thr Ala GlyGln Glu Gly Leu Gln Tyr Gln Pro Gly Asp His 785 790 795 800 Ile Gly IleCys Pro Pro Asn Arg Pro Gly Leu Val Glu Ala Leu Leu 805 810 815 Ser ArgVal Glu Asp Pro Pro Pro Pro Thr Glu Ser Val Ala Val Glu 820 825 830 GlnLeu Glu Lys Gly Ser Pro Gly Gly Pro Pro Pro Ser Trp Val Arg 835 840 845Asp Pro Arg Leu Pro Pro Cys Thr Leu Arg Gln Ala Leu Thr Phe Phe 850 855860 Leu Asp Ile Thr Ser Pro Pro Ser Pro Arg Leu Leu Arg Leu Leu Ser 865870 875 880 Thr Leu Ala Glu Glu Pro Ser Glu Gln Gln Glu Leu Glu Thr LeuSer 885 890 895 Gln Asp Pro Arg Arg Tyr Glu Glu Trp Lys Trp Phe Arg CysPro Thr 900 905 910 Leu Leu Glu Val Leu Glu Gln Phe Pro Ser Val Ala LeuPro Ala Pro 915 920 925 Leu Leu Leu Thr Gln Leu Pro Leu Leu Gln Pro ArgTyr Tyr Ser Val 930 935 940 Ser Ser Ala Pro Asn Ala His Pro Gly Glu ValHis Leu Thr Val Ala 945 950 955 960 Val Leu Ala Tyr Arg Thr Gln Asp GlyLeu Gly Pro Leu His Tyr Gly 965 970 975 Val Cys Ser Thr Trp Leu Ser GlnLeu Lys Thr Gly Asp Pro Val Pro 980 985 990 Cys Phe Ile Arg Gly Ala ProSer Phe Arg Leu Pro Pro Asp Pro Tyr 995 1000 1005 Val Pro Cys Ile LeuVal Gly Pro Gly Thr Gly Ile Ala Pro Phe 1010 1015 1020 Arg Gly Phe TrpGln Glu Arg Leu His Asp Ile Glu Ser Lys Gly 1025 1030 1035 Leu Gln ProAla Pro Met Thr Leu Val Phe Gly Cys Arg Cys Ser 1040 1045 1050 Gln LeuAsp His Leu Tyr Arg Asp Glu Val Gln Asp Ala Gln Glu 1055 1060 1065 ArgGly Val Phe Gly Arg Val Leu Thr Ala Phe Ser Arg Glu Pro 1070 1075 1080Asp Ser Pro Lys Thr Tyr Val Gln Asp Ile Leu Arg Thr Glu Leu 1085 10901095 Ala Ala Glu Val His Arg Val Leu Cys Leu Glu Arg Gly His Met 11001105 1110 Phe Val Cys Gly Asp Val Thr Met Ala Thr Ser Val Leu Gln Thr1115 1120 1125 Val Gln Arg Ile Leu Ala Thr Glu Gly Asp Met Glu Leu AspGlu 1130 1135 1140 Ala Gly Asp Val Ile Gly Val Leu Arg Asp Gln Gln ArgTyr His 1145 1150 1155 Glu Asp Ile Phe Gly Leu Thr Leu Arg Thr Gln GluVal Thr Ser 1160 1165 1170 Arg Ile Arg Thr Gln Ser Phe Ser Leu Gln GluArg His Leu Arg 1175 1180 1185 Gly Ala Val Pro Trp Ala Phe Asp Pro ProGly Pro Asp Thr Pro 1190 1195 1200 Gly Pro 1205 5 1203 PRT Homo sapiensMUTAGEN (1177)..(1177) S is substituted 5 Met Gly Asn Leu Lys Ser ValAla Gln Glu Pro Gly Pro Pro Cys Gly 1 5 10 15 Leu Gly Leu Gly Leu GlyLeu Gly Leu Cys Gly Lys Gln Gly Pro Ala 20 25 30 Thr Pro Ala Pro Glu ProSer Arg Ala Pro Ala Ser Leu Leu Pro Pro 35 40 45 Ala Pro Glu His Ser ProPro Ser Ser Pro Leu Thr Gln Pro Pro Glu 50 55 60 Gly Pro Lys Phe Pro ArgVal Lys Asn Trp Glu Val Gly Ser Ile Thr 65 70 75 80 Tyr Asp Thr Leu SerAla Gln Ala Gln Gln Asp Gly Pro Cys Thr Pro 85 90 95 Arg Arg Cys Leu GlySer Leu Val Phe Pro Arg Lys Leu Gln Gly Arg 100 105 110 Pro Ser Pro GlyPro Pro Ala Pro Glu Gln Leu Leu Ser Gln Ala Arg 115 120 125 Asp Phe IleAsn Gln Tyr Tyr Ser Ser Ile Lys Arg Ser Gly Ser Gln 130 135 140 Ala HisGlu Gln Arg Leu Gln Glu Val Glu Ala Glu Val Ala Ala Thr 145 150 155 160Gly Thr Tyr Gln Leu Arg Glu Ser Glu Leu Val Phe Gly Ala Lys Gln 165 170175 Ala Trp Arg Asn Ala Pro Arg Cys Val Gly Arg Ile Gln Trp Gly Lys 180185 190 Leu Gln Val Phe Asp Ala Arg Asp Cys Arg Ser Ala Gln Glu Met Phe195 200 205 Thr Tyr Ile Cys Asn His Ile Lys Tyr Ala Thr Asn Arg Gly AsnLeu 210 215 220 Arg Ser Ala Ile Thr Val Phe Pro Gln Arg Cys Pro Gly ArgGly Asp 225 230 235 240 Phe Arg Ile Trp Asn Ser Gln Leu Val Arg Tyr AlaGly Tyr Arg Gln 245 250 255 Gln Asp Gly Ser Val Arg Gly Asp Pro Ala AsnVal Glu Ile Thr Glu 260 265 270 Leu Cys Ile Gln His Gly Trp Thr Pro GlyAsn Gly Arg Phe Asp Val 275 280 285 Leu Pro Leu Leu Leu Gln Ala Pro AspGlu Pro Pro Glu Leu Phe Leu 290 295 300 Leu Pro Pro Glu Leu Val Leu GluVal Pro Leu Glu His Pro Thr Leu 305 310 315 320 Glu Trp Phe Ala Ala LeuGly Leu Arg Trp Tyr Ala Leu Pro Ala Val 325 330 335 Ser Asn Met Leu LeuGlu Ile Gly Gly Leu Glu Phe Pro Ala Ala Pro 340 345 350 Phe Ser Gly TrpTyr Met Ser Thr Glu Ile Gly Thr Arg Asn Leu Cys 355 360 365 Asp Pro HisArg Tyr Asn Ile Leu Glu Asp Val Ala Val Cys Met Asp 370 375 380 Leu AspThr Arg Thr Thr Ser Ser Leu Trp Lys Asp Lys Ala Ala Val 385 390 395 400Glu Ile Asn Val Ala Val Leu His Ser Tyr Gln Leu Ala Lys Val Thr 405 410415 Ile Val Asp His His Ala Ala Thr Ala Ser Phe Met Lys His Leu Glu 420425 430 Asn Glu Gln Lys Ala Arg Gly Gly Cys Pro Ala Asp Trp Ala Trp Ile435 440 445 Val Pro Pro Ile Ser Gly Ser Leu Thr Pro Val Phe His Gln GluMet 450 455 460 Val Asn Tyr Phe Leu Ser Pro Ala Phe Arg Tyr Gln Pro AspPro Trp 465 470 475 480 Lys Gly Ser Ala Ala Lys Gly Thr Gly Ile Thr ArgLys Lys Thr Phe 485 490 495 Lys Glu Val Ala Asn Ala Val Lys Ile Ser AlaSer Leu Met Gly Thr 500 505 510 Val Met Ala Lys Arg Val Lys Ala Thr IleLeu Tyr Gly Ser Glu Thr 515 520 525 Gly Arg Ala Gln Ser Tyr Ala Gln GlnLeu Gly Arg Leu Phe Arg Lys 530 535 540 Ala Phe Asp Pro Arg Val Leu CysMet Asp Glu Tyr Asp Val Val Ser 545 550 555 560 Leu Glu His Glu Thr LeuVal Leu Val Val Thr Ser Thr Phe Gly Asn 565 570 575 Gly Asp Pro Pro GluAsn Gly Glu Ser Phe Ala Ala Ala Leu Met Glu 580 585 590 Met Ser Gly ProTyr Asn Ser Ser Pro Arg Pro Glu Gln His Lys Ser 595 600 605 Tyr Lys IleArg Phe Asn Ser Ile Ser Cys Ser Asp Pro Leu Val Ser 610 615 620 Ser TrpArg Arg Lys Arg Lys Glu Ser Ser Asn Thr Asp Ser Ala Gly 625 630 635 640Ala Leu Gly Thr Leu Arg Phe Cys Val Phe Gly Leu Gly Ser Arg Ala 645 650655 Tyr Pro His Phe Cys Ala Phe Ala Arg Ala Val Asp Thr Arg Leu Glu 660665 670 Glu Leu Gly Gly Glu Arg Leu Leu Gln Leu Gly Gln Gly Asp Glu Leu675 680 685 Cys Gly Gln Glu Glu Ala Phe Arg Gly Trp Ala Gln Ala Ala PheGln 690 695 700 Ala Ala Cys Glu Thr Phe Cys Val Gly Glu Asp Ala Lys AlaAla Ala 705 710 715 720 Arg Asp Ile Phe Ser Pro Lys Arg Ser Trp Lys ArgGln Arg Tyr Arg 725 730 735 Leu Ser Ala Gln Ala Glu Gly Leu Gln Leu LeuPro Gly Leu Ile His 740 745 750 Val His Arg Arg Lys Met Phe Gln Ala ThrIle Arg Ser Val Glu Asn 755 760 765 Leu Gln Ser Ser Lys Ser Thr Arg AlaThr Ile Leu Val Arg Leu Asp 770 775 780 Thr Gly Gly Gln Glu Gly Leu GlnTyr Gln Pro Gly Asp His Ile Gly 785 790 795 800 Val Cys Pro Pro Asn ArgPro Gly Leu Val Glu Ala Leu Leu Ser Arg 805 810 815 Val Glu Asp Pro ProAla Pro Thr Glu Pro Val Ala Val Glu Gln Leu 820 825 830 Glu Lys Gly SerPro Gly Gly Pro Pro Pro Gly Trp Val Arg Asp Pro 835 840 845 Arg Leu ProPro Cys Thr Leu Arg Gln Ala Leu Thr Phe Phe Leu Asp 850 855 860 Ile ThrSer Pro Pro Ser Pro Gln Leu Leu Arg Leu Leu Ser Thr Leu 865 870 875 880Ala Glu Glu Pro Arg Glu Gln Gln Glu Leu Glu Ala Leu Ser Gln Asp 885 890895 Pro Arg Arg Tyr Glu Glu Trp Lys Trp Phe Arg Cys Pro Thr Leu Leu 900905 910 Glu Val Leu Glu Gln Phe Pro Ser Val Ala Leu Pro Ala Pro Leu Leu915 920 925 Leu Thr Gln Leu Pro Leu Leu Gln Pro Arg Tyr Tyr Ser Val SerSer 930 935 940 Ala Pro Ser Thr His Pro Gly Glu Ile His Leu Thr Val AlaVal Leu 945 950 955 960 Ala Tyr Arg Thr Gln Asp Gly Leu Gly Pro Leu HisTyr Gly Val Cys 965 970 975 Ser Thr Trp Leu Ser Gln Leu Lys Pro Gly AspPro Val Pro Cys Phe 980 985 990 Ile Arg Gly Ala Pro Ser Phe Arg Leu ProPro Asp Pro Ser Leu Pro 995 1000 1005 Cys Ile Leu Val Gly Pro Gly ThrGly Ile Ala Pro Phe Arg Gly 1010 1015 1020 Phe Trp Gln Glu Arg Leu HisAsp Ile Glu Ser Lys Gly Leu Gln 1025 1030 1035 Pro Thr Pro Met Thr LeuVal Phe Gly Cys Arg Cys Ser Gln Leu 1040 1045 1050 Asp His Leu Tyr ArgAsp Glu Val Gln Asn Ala Gln Gln Arg Gly 1055 1060 1065 Val Phe Gly ArgVal Leu Thr Ala Phe Ser Arg Glu Pro Asp Asn 1070 1075 1080 Pro Lys ThrTyr Val Gln Asp Ile Leu Arg Thr Glu Leu Ala Ala 1085 1090 1095 Glu ValHis Arg Val Leu Cys Leu Glu Arg Gly His Met Phe Val 1100 1105 1110 CysGly Asp Val Thr Met Ala Thr Asn Val Leu Gln Thr Val Gln 1115 1120 1125Arg Ile Leu Ala Thr Glu Gly Asp Met Glu Leu Asp Glu Ala Gly 1130 11351140 Asp Val Ile Gly Val Leu Arg Asp Gln Gln Arg Tyr His Glu Asp 11451150 1155 Ile Phe Gly Leu Thr Leu Arg Thr Gln Glu Val Thr Ser Arg Ile1160 1165 1170 Arg Thr Gln Ser Phe Ser Leu Gln Glu Arg Gln Leu Arg GlyAla 1175 1180 1185 Val Pro Trp Ala Phe Asp Pro Pro Gly Ser Asp Thr AsnSer Pro 1190 1195 1200 6 1203 PRT Homo sapiens 6 Met Gly Asn Leu Lys SerVal Ala Gln Glu Pro Gly Pro Pro Cys Gly 1 5 10 15 Leu Gly Leu Gly LeuGly Leu Gly Leu Cys Gly Lys Gln Gly Pro Ala 20 25 30 Thr Pro Ala Pro GluPro Ser Arg Ala Pro Ala Ser Leu Leu Pro Pro 35 40 45 Ala Pro Glu His SerPro Pro Ser Ser Pro Leu Thr Gln Pro Pro Glu 50 55 60 Gly Pro Lys Phe ProArg Val Lys Asn Trp Glu Val Gly Ser Ile Thr 65 70 75 80 Tyr Asp Thr LeuSer Ala Gln Ala Gln Gln Asp Gly Pro Cys Thr Pro 85 90 95 Arg Arg Cys LeuGly Ser Leu Val Phe Pro Arg Lys Leu Gln Gly Arg 100 105 110 Pro Ser ProGly Pro Pro Ala Pro Glu Gln Leu Leu Ser Gln Ala Arg 115 120 125 Asp PheIle Asn Gln Tyr Tyr Ser Ser Ile Lys Arg Ser Gly Ser Gln 130 135 140 AlaHis Glu Gln Arg Leu Gln Glu Val Glu Ala Glu Val Ala Ala Thr 145 150 155160 Gly Thr Tyr Gln Leu Arg Glu Ser Glu Leu Val Phe Gly Ala Lys Gln 165170 175 Ala Trp Arg Asn Ala Pro Arg Cys Val Gly Arg Ile Gln Trp Gly Lys180 185 190 Leu Gln Val Phe Asp Ala Arg Asp Cys Arg Ser Ala Gln Glu MetPhe 195 200 205 Thr Tyr Ile Cys Asn His Ile Lys Tyr Ala Thr Asn Arg GlyAsn Leu 210 215 220 Arg Ser Ala Ile Thr Val Phe Pro Gln Arg Cys Pro GlyArg Gly Asp 225 230 235 240 Phe Arg Ile Trp Asn Ser Gln Leu Val Arg TyrAla Gly Tyr Arg Gln 245 250 255 Gln Asp Gly Ser Val Arg Gly Asp Pro AlaAsn Val Glu Ile Thr Glu 260 265 270 Leu Cys Ile Gln His Gly Trp Thr ProGly Asn Gly Arg Phe Asp Val 275 280 285 Leu Pro Leu Leu Leu Gln Ala ProAsp Glu Pro Pro Glu Leu Phe Leu 290 295 300 Leu Pro Pro Glu Leu Val LeuGlu Val Pro Leu Glu His Pro Thr Leu 305 310 315 320 Glu Trp Phe Ala AlaLeu Gly Leu Arg Trp Tyr Ala Leu Pro Ala Val 325 330 335 Ser Asn Met LeuLeu Glu Ile Gly Gly Leu Glu Phe Pro Ala Ala Pro 340 345 350 Phe Ser GlyTrp Tyr Met Ser Thr Glu Ile Gly Thr Arg Asn Leu Cys 355 360 365 Asp ProHis Arg Tyr Asn Ile Leu Glu Asp Val Ala Val Cys Met Asp 370 375 380 LeuAsp Thr Arg Thr Thr Ser Ser Leu Trp Lys Asp Lys Ala Ala Val 385 390 395400 Glu Ile Asn Val Ala Val Leu His Ser Tyr Gln Leu Ala Lys Val Thr 405410 415 Ile Val Asp His His Ala Ala Thr Ala Ser Phe Met Lys His Leu Glu420 425 430 Asn Glu Gln Lys Ala Arg Gly Gly Cys Pro Ala Asp Trp Ala TrpIle 435 440 445 Val Pro Pro Ile Ser Gly Ser Leu Thr Pro Val Phe His GlnGlu Met 450 455 460 Val Asn Tyr Phe Leu Ser Pro Ala Phe Arg Tyr Gln ProAsp Pro Trp 465 470 475 480 Lys Gly Ser Ala Ala Lys Gly Thr Gly Ile ThrArg Lys Lys Thr Phe 485 490 495 Lys Glu Val Ala Asn Ala Val Lys Ile SerAla Ser Leu Met Gly Thr 500 505 510 Val Met Ala Lys Arg Val Lys Ala ThrIle Leu Tyr Gly Ser Glu Thr 515 520 525 Gly Arg Ala Gln Ser Tyr Ala GlnGln Leu Gly Arg Leu Phe Arg Lys 530 535 540 Ala Phe Asp Pro Arg Val LeuCys Met Asp Glu Tyr Asp Val Val Ser 545 550 555 560 Leu Glu His Glu ThrLeu Val Leu Val Val Thr Ser Thr Phe Gly Asn 565 570 575 Gly Asp Pro ProGlu Asn Gly Glu Ser Phe Ala Ala Ala Leu Met Glu 580 585 590 Met Ser GlyPro Tyr Asn Ser Ser Pro Arg Pro Glu Gln His Lys Ser 595 600 605 Tyr LysIle Arg Phe Asn Ser Ile Ser Cys Ser Asp Pro Leu Val Ser 610 615 620 SerTrp Arg Arg Lys Arg Lys Glu Ser Ser Asn Thr Asp Ser Ala Gly 625 630 635640 Ala Leu Gly Thr Leu Arg Phe Cys Val Phe Gly Leu Gly Ser Arg Ala 645650 655 Tyr Pro His Phe Cys Ala Phe Ala Arg Ala Val Asp Thr Arg Leu Glu660 665 670 Glu Leu Gly Gly Glu Arg Leu Leu Gln Leu Gly Gln Gly Asp GluLeu 675 680 685 Cys Gly Gln Glu Glu Ala Phe Arg Gly Trp Ala Gln Ala AlaPhe Gln 690 695 700 Ala Ala Cys Glu Thr Phe Cys Val Gly Glu Asp Ala LysAla Ala Ala 705 710 715 720 Arg Asp Ile Phe Ser Pro Lys Arg Ser Trp LysArg Gln Arg Tyr Arg 725 730 735 Leu Ser Ala Gln Ala Glu Gly Leu Gln LeuLeu Pro Gly Leu Ile His 740 745 750 Val His Arg Arg Lys Met Phe Gln AlaThr Ile Arg Ser Val Glu Asn 755 760 765 Leu Gln Ser Ser Lys Ser Thr ArgAla Thr Ile Leu Val Arg Leu Asp 770 775 780 Thr Gly Gly Gln Glu Gly LeuGln Tyr Gln Pro Gly Asp His Ile Gly 785 790 795 800 Val Cys Pro Pro AsnArg Pro Gly Leu Val Glu Ala Leu Leu Ser Arg 805 810 815 Val Glu Asp ProPro Ala Pro Thr Glu Pro Val Ala Val Glu Gln Leu 820 825 830 Glu Lys GlySer Pro Gly Gly Pro Pro Pro Gly Trp Val Arg Asp Pro 835 840 845 Arg LeuPro Pro Cys Thr Leu Arg Gln Ala Leu Thr Phe Phe Leu Asp 850 855 860 IleThr Ser Pro Pro Ser Pro Gln Leu Leu Arg Leu Leu Ser Thr Leu 865 870 875880 Ala Glu Glu Pro Arg Glu Gln Gln Glu Leu Glu Ala Leu Ser Gln Asp 885890 895 Pro Arg Arg Tyr Glu Glu Trp Lys Trp Phe Arg Cys Pro Thr Leu Leu900 905 910 Glu Val Leu Glu Gln Phe Pro Ser Val Ala Leu Pro Ala Pro LeuLeu 915 920 925 Leu Thr Gln Leu Pro Leu Leu Gln Pro Arg Tyr Tyr Ser ValSer Ser 930 935 940 Ala Pro Ser Thr His Pro Gly Glu Ile His Leu Thr ValAla Val Leu 945 950 955 960 Ala Tyr Arg Thr Gln Asp Gly Leu Gly Pro LeuHis Tyr Gly Val Cys 965 970 975 Ser Thr Trp Leu Ser Gln Leu Lys Pro GlyAsp Pro Val Pro Cys Phe 980 985 990 Ile Arg Gly Ala Pro Ser Phe Arg LeuPro Pro Asp Pro Ser Leu Pro 995 1000 1005 Cys Ile Leu Val Gly Pro GlyThr Gly Ile Ala Pro Phe Arg Gly 1010 1015 1020 Phe Trp Gln Glu Arg LeuHis Asp Ile Glu Ser Lys Gly Leu Gln 1025 1030 1035 Pro Thr Pro Met ThrLeu Val Phe Gly Cys Arg Cys Ser Gln Leu 1040 1045 1050 Asp His Leu TyrArg Asp Glu Val Gln Asn Ala Gln Gln Arg Gly 1055 1060 1065 Val Phe GlyArg Val Leu Thr Ala Phe Ser Arg Glu Pro Asp Asn 1070 1075 1080 Pro LysThr Tyr Val Gln Asp Ile Leu Arg Thr Glu Leu Ala Ala 1085 1090 1095 GluVal His Arg Val Leu Cys Leu Glu Arg Gly His Met Phe Val 1100 1105 1110Cys Gly Asp Val Thr Met Ala Thr Asn Val Leu Gln Thr Val Gln 1115 11201125 Arg Ile Leu Ala Thr Glu Gly Asp Met Glu Leu Asp Glu Ala Gly 11301135 1140 Asp Val Ile Gly Val Leu Arg Asp Gln Gln Arg Tyr His Glu Asp1145 1150 1155 Ile Phe Gly Leu Thr Leu Arg Thr Gln Glu Val Thr Ser ArgIle 1160 1165 1170 Arg Thr Gln Asp Phe Ser Leu Gln Glu Arg Gln Leu ArgGly Ala 1175 1180 1185 Val Pro Trp Ala Phe Asp Pro Pro Gly Ser Asp ThrAsn Ser Pro 1190 1195 1200 7 1202 PRT Homo sapiens 7 Met Gly Asn Leu LysSer Val Ala Gln Glu Pro Gly Pro Pro Cys Gly 1 5 10 15 Leu Gly Leu GlyLeu Gly Leu Gly Leu Cys Gly Lys Gln Gly Pro Ala 20 25 30 Thr Pro Ala ProGlu Pro Ser Arg Ala Pro Ala Ser Leu Leu Pro Pro 35 40 45 Ala Pro Glu HisSer Pro Pro Ser Ser Pro Leu Thr Gln Pro Pro Glu 50 55 60 Gly Pro Lys PhePro Arg Val Lys Asn Trp Glu Val Gly Ser Ile Thr 65 70 75 80 Tyr Asp ThrLeu Ser Ala Gln Ala Gln Gln Asp Gly Pro Cys Thr Pro 85 90 95 Arg Arg CysLeu Gly Ser Leu Val Phe Pro Arg Lys Leu Gln Gly Arg 100 105 110 Pro SerPro Gly Pro Pro Ala Pro Glu Gln Leu Leu Ser Gln Ala Arg 115 120 125 AspPhe Ile Asn Gln Tyr Tyr Ser Ser Ile Lys Arg Ser Gly Ser Gln 130 135 140Ala His Glu Gln Arg Leu Gln Glu Val Glu Ala Glu Val Ala Ala Thr 145 150155 160 Gly Thr Tyr Gln Leu Arg Glu Ser Glu Leu Val Phe Gly Ala Lys Gln165 170 175 Ala Trp Arg Asn Ala Pro Arg Cys Val Gly Arg Ile Gln Trp GlyLys 180 185 190 Leu Gln Val Phe Asp Ala Arg Asp Cys Arg Ser Ala Gln GluMet Phe 195 200 205 Thr Tyr Ile Cys Asn His Ile Lys Tyr Ala Thr Asn ArgGly Asn Leu 210 215 220 Arg Ser Ala Ile Thr Val Phe Pro Gln Arg Cys ProGly Arg Gly Asp 225 230 235 240 Phe Arg Ile Trp Asn Ser Gln Leu Val ArgTyr Ala Gly Tyr Arg Gln 245 250 255 Gln Asp Gly Ser Val Arg Gly Asp ProAla Asn Val Glu Ile Thr Glu 260 265 270 Leu Cys Ile Gln His Gly Trp ThrPro Gly Asn Gly Arg Phe Asp Val 275 280 285 Leu Pro Leu Leu Leu Gln AlaPro Asp Glu Pro Pro Glu Leu Phe Leu 290 295 300 Leu Pro Pro Glu Leu ValLeu Glu Val Pro Leu Glu His Pro Thr Leu 305 310 315 320 Glu Trp Phe AlaAla Leu Gly Leu Arg Trp Tyr Ala Leu Pro Ala Val 325 330 335 Ser Asn MetLeu Leu Glu Ile Gly Gly Leu Glu Phe Pro Ala Ala Pro 340 345 350 Phe SerGly Trp Tyr Met Ser Thr Glu Ile Gly Thr Arg Asn Leu Cys 355 360 365 AspPro His Arg Tyr Asn Ile Leu Glu Asp Val Ala Val Cys Met Asp 370 375 380Leu Asp Thr Arg Thr Thr Ser Ser Leu Trp Lys Asp Lys Ala Ala Val 385 390395 400 Glu Ile Asn Val Ala Val Leu His Ser Tyr Gln Leu Ala Lys Val Thr405 410 415 Ile Val Asp His His Ala Ala Thr Ala Ser Phe Met Lys His LeuGlu 420 425 430 Asn Glu Gln Lys Ala Arg Gly Gly Cys Pro Ala Asp Trp AlaTrp Ile 435 440 445 Val Pro Pro Ile Ser Gly Ser Leu Thr Pro Val Phe HisGln Glu Met 450 455 460 Val Asn Tyr Phe Leu Ser Pro Ala Phe Arg Tyr GlnPro Asp Pro Trp 465 470 475 480 Lys Gly Ser Ala Ala Lys Gly Thr Gly IleThr Arg Lys Lys Thr Phe 485 490 495 Lys Glu Val Ala Asn Ala Val Lys IleSer Ala Ser Leu Met Gly Thr 500 505 510 Val Met Ala Lys Arg Val Lys AlaThr Ile Leu Tyr Gly Ser Glu Thr 515 520 525 Gly Arg Ala Gln Ser Tyr AlaGln Gln Leu Gly Arg Leu Phe Arg Lys 530 535 540 Ala Phe Asp Pro Arg ValLeu Cys Met Asp Glu Tyr Asp Val Val Ser 545 550 555 560 Leu Glu His GluThr Leu Val Leu Val Val Thr Ser Thr Phe Gly Asn 565 570 575 Gly Asp ProPro Glu Asn Gly Glu Ser Phe Ala Ala Ala Leu Met Glu 580 585 590 Met SerGly Pro Tyr Asn Ser Ser Pro Arg Pro Glu Gln His Lys Ser 595 600 605 TyrLys Ile Arg Phe Asn Ser Ile Ser Cys Ser Asp Pro Leu Val Ser 610 615 620Ser Trp Arg Arg Lys Arg Lys Glu Ser Ser Asn Thr Asp Ser Ala Gly 625 630635 640 Ala Leu Gly Thr Leu Arg Phe Cys Val Phe Gly Leu Gly Ser Arg Ala645 650 655 Tyr Pro His Phe Cys Ala Phe Ala Arg Ala Val Asp Thr Arg LeuGlu 660 665 670 Glu Leu Gly Gly Glu Arg Leu Leu Gln Leu Gly Gln Gly AspGlu Leu 675 680 685 Cys Gly Gln Glu Glu Ala Phe Arg Gly Trp Ala Gln AlaAla Phe Gln 690 695 700 Ala Ala Cys Glu Thr Phe Cys Val Gly Glu Asp AlaLys Ala Ala Ala 705 710 715 720 Arg Asp Ile Phe Ser Pro Lys Arg Ser TrpLys Arg Gln Arg Tyr Arg 725 730 735 Leu Ser Ala Gln Ala Glu Gly Leu GlnLeu Leu Pro Gly Leu Ile His 740 745 750 Val His Arg Arg Lys Met Phe GlnAla Thr Ile Arg Ser Val Glu Asn 755 760 765 Leu Gln Ser Ser Lys Ser ThrArg Ala Thr Ile Leu Val Arg Leu Asp 770 775 780 Thr Gly Gly Gln Glu GlyLeu Gln Tyr Gln Pro Gly Asp His Ile Gly 785 790 795 800 Val Cys Pro ProAsn Arg Pro Gly Leu Val Glu Ala Leu Leu Ser Arg 805 810 815 Val Glu AspPro Pro Ala Pro Thr Glu Pro Val Ala Val Glu Gln Leu 820 825 830 Glu LysGly Ser Pro Gly Gly Pro Pro Pro Gly Trp Val Arg Asp Pro 835 840 845 ArgLeu Pro Pro Cys Thr Leu Arg Gln Ala Leu Thr Phe Phe Leu Asp 850 855 860Ile Thr Ser Pro Pro Ser Pro Gln Leu Leu Arg Leu Leu Ser Thr Leu 865 870875 880 Ala Glu Glu Pro Arg Glu Gln Gln Glu Leu Glu Ala Leu Ser Gln Asp885 890 895 Pro Arg Arg Tyr Glu Glu Trp Lys Trp Phe Arg Cys Pro Thr LeuLeu 900 905 910 Glu Val Leu Glu Gln Phe Pro Ser Val Ala Leu Pro Ala ProLeu Leu 915 920 925 Leu Thr Gln Leu Pro Leu Leu Gln Pro Arg Tyr Tyr SerVal Ser Ser 930 935 940 Ala Pro Ser Thr His Pro Gly Glu Ile His Leu ThrVal Ala Val Leu 945 950 955 960 Ala Tyr Arg Thr Gln Asp Gly Leu Gly ProLeu His Tyr Gly Val Cys 965 970 975 Ser Thr Trp Leu Ser Gln Leu Lys ProGly Asp Pro Val Pro Cys Phe 980 985 990 Ile Arg Gly Ala Pro Ser Phe ArgLeu Pro Pro Asp Pro Ser Leu Pro 995 1000 1005 Cys Ile Leu Val Gly ProGly Thr Gly Ile Ala Pro Phe Arg Gly 1010 1015 1020 Phe Trp Gln Glu ArgLeu His Asp Ile Glu Ser Lys Gly Leu Gln 1025 1030 1035 Pro Thr Pro MetThr Leu Val Phe Gly Cys Arg Cys Ser Gln Leu 1040 1045 1050 Asp His LeuTyr Arg Asp Glu Val Gln Asn Ala Gln Gln Arg Gly 1055 1060 1065 Val PheGly Arg Val Leu Thr Ala Phe Ser Arg Glu Pro Asp Asn 1070 1075 1080 ProLys Thr Tyr Val Gln Asp Ile Leu Arg Thr Glu Leu Ala Ala 1085 1090 1095Glu Val His Arg Val Leu Cys Leu Glu Arg Gly His Met Phe Val 1100 11051110 Cys Gly Asp Val Thr Met Ala Thr Asn Val Leu Gln Thr Val Gln 11151120 1125 Arg Ile Leu Ala Thr Glu Gly Asp Met Glu Leu Asp Glu Ala Gly1130 1135 1140 Asp Val Ile Gly Val Leu Arg Asp Gln Gln Arg Tyr His GluAsp 1145 1150 1155 Ile Phe Gly Leu Thr Leu Arg Thr Gln Glu Val Thr SerArg Ile 1160 1165 1170 Arg Thr Gln Glu Ser Leu Gln Glu Arg Gln Leu ArgGly Ala Val 1175 1180 1185 Pro Trp Ala Phe Asp Pro Pro Gly Ser Asp ThrAsn Ser Pro 1190 1195 1200 8 3612 DNA Homo sapiens 8 atgggcaacttgaagagcgt ggcccaggag cctgggccac cctgcggcct ggggctgggg 60 ctgggccttgggctgtgcgg caagcagggc ccagccaccc cggcccctga gcccagccgg 120 gccccagcatccctactccc accagcgcca gaacacagcc ccccgagctc cccgctaacc 180 cagcccccagaggggcccaa gttccctcgt gtgaagaact gggaggtggg gagcatcacc 240 tatgacaccctcagcgccca ggcgcagcag gatgggccct gcaccccaag acgctgcctg 300 ggctccctggtatttccacg gaaactacag ggccggccct cccccggccc cccggcccct 360 gagcagctgctgagtcaggc ccgggacttc atcaaccagt actacagctc cattaagagg 420 agcggctcccaggcccacga acagcggctt caagaggtgg aagccgaggt ggcagccaca 480 ggcacctaccagcttaggga gagcgagctg gtgttcgggg ctaagcaggc ctggcgcaac 540 gctccccgctgcgtgggccg gatccagtgg gggaagctgc aggtgttcga tgcccgggac 600 tgcaggtctgcacaggaaat gttcacctac atctgcaacc acatcaagta tgccaccaac 660 cggggcaaccttcgctcggc catcacagtg ttcccgcagc gctgccctgg ccgaggagac 720 ttccgaatctggaacagcca gctggtgcgc tacgcgggct accggcagca ggacggctct 780 gtgcggggggacccagccaa cgtggagatc accgagctct gcattcagca cggctggacc 840 ccaggaaacggtcgcttcga cgtgctgccc ctgctgctgc aggccccaga tgagccccca 900 gaactcttccttctgccccc cgagctggtc cttgaggtgc ccctggagca ccccacgctg 960 gagtggtttgcagccctggg cctgcgctgg tacgccctcc cggcagtgtc caacatgctg 1020 ctggaaattgggggcctgga gttccccgca gcccccttca gtggctggta catgagcact 1080 gagatcggcacgaggaacct gtgtgaccct caccgctaca acatcctgga ggatgtggct 1140 gtctgcatggacctggatac ccggaccacc tcgtccctgt ggaaagacaa ggcagcagtg 1200 gaaatcaacgtggccgtgct gcacagttac cagctagcca aagtcaccat cgtggaccac 1260 cacgccgccacggcctcttt catgaagcac ctggagaatg agcagaaggc cagggggggc 1320 tgccctgcagactgggcctg gatcgtgccc cccatctcgg gcagcctcac tcctgttttc 1380 catcaggagatggtcaacta tttcctgtcc ccggccttcc gctaccagcc agacccctgg 1440 aaggggagtgccgccaaggg caccggcatc accaggaaga agacctttaa agaagtggcc 1500 aacgccgtgaagatctccgc ctcgctcatg ggcacggtga tggcgaagcg agtgaaggcg 1560 acaatcctgtatggctccga gaccggccgg gcccagagct acgcacagca gctggggaga 1620 ctcttccggaaggcttttga tccccgggtc ctgtgtatgg atgagtatga cgtggtgtcc 1680 ctcgaacacgagacgctggt gctggtggta accagcacat ttgggaatgg ggatcccccg 1740 gagaatggagagagctttgc agctgccctg atggagatgt ccggccccta caacagctcc 1800 cctcggccggaacagcacaa gagttataag atccgcttca acagcatctc ctgctcagac 1860 ccactggtgtcctcttggcg gcggaagagg aaggagtcca gtaacacaga cagtgcaggg 1920 gccctgggcaccctcaggtt ctgtgtgttc gggctcggct cccgggcata cccccacttc 1980 tgcgcctttgctcgtgccgt ggacacacgg ctggaggaac tgggcgggga gcggctgctg 2040 cagctgggccagggcgacga gctgtgcggc caggaggagg ccttccgagg ctgggcccag 2100 gctgccttccaggccgcctg tgagaccttc tgtgtgggag aggatgccaa ggccgccgcc 2160 cgagacatcttcagccccaa acggagctgg aagcgccaga ggtaccggct gagcgcccag 2220 gccgagggcctgcagttgct gccaggtctg atccacgtgc acaggcggaa gatgttccag 2280 gctacaatccgctcagtgga aaacctgcaa agcagcaagt ccacgagggc caccatcctg 2340 gtgcgcctggacaccggagg ccaggagggg ctgcagtacc agccggggga ccacataggt 2400 gtctgcccgcccaaccggcc cggccttgtg gaggcgctgc tgagccgcgt ggaggacccg 2460 ccggcgcccactgagcccgt ggcagtagag cagctggaga agggcagccc tggtggccct 2520 ccccccggctgggtgcggga cccccggctg cccccgtgca cgctgcgcca ggctctcacc 2580 ttcttcctggacatcacctc cccacccagc cctcagctct tgcggctgct cagcaccttg 2640 gcagaagagcccagggaaca gcaggagctg gaggccctca gccaggatcc ccgacgctac 2700 gaggagtggaagtggttccg ctgccccacg ctgctggagg tgctggagca gttcccgtcg 2760 gtggcgctgcctgccccact gctcctcacc cagctgcctc tgctccagcc ccggtactac 2820 tcagtcagctcggcacccag cacccaccca ggagagatcc acctcactgt agctgtgctg 2880 gcatacaggactcaggatgg gctgggcccc ctgcactatg gagtctgctc cacgtggcta 2940 agccagctcaagcccggaga ccctgtgccc tgcttcatcc ggggggctcc ctccttccgg 3000 ctgccacccgatcccagctt gccctgcatc ctggtgggtc caggcactgg cattgccccc 3060 ttccggggattctggcagga gcggctgcat gacattgaga gcaaagggct gcagcccact 3120 cccatgactttggtgttcgg ctgccgatgc tcccaacttg accatctcta ccgcgacgag 3180 gtgcagaacgcccagcagcg cggggtgttt ggccgagtcc tcaccgcctt ctcccgggaa 3240 cctgacaaccccaagaccta cgtgcaggac atcctgagga cggagctggc tgcggaggtg 3300 caccgcgtgctgtgcctcga gcggggccac atgtttgtct gcggcgatgt taccatggca 3360 accaacgtcctgcagaccgt gcagcgcatc ctggcgacgg agggcgacat ggagctggac 3420 gaggccggcgacgtcatcgg cgtgctgcgg gatcagcaac gctaccacga agacattttc 3480 gggctcacgctgcgcaccca ggaggtgaca agccgcatac gcacccagag cttttccttg 3540 caggagcgtcagttgcgggg cgcagtgccc tgggcgttcg accctcccgg ctcagacacc 3600 aacagcccctga 3612 9 4077 DNA Homo sapiens 9 gaattcccac tctgctgcct gctccagcagacggacgcac agtaacatgg gcaacttgaa 60 gagcgtggcc caggagcctg ggccaccctgcggcctgggg ctggggctgg gccttgggct 120 gtgcggcaag cagggcccag ccaccccggcccctgagccc agccgggccc cagcatccct 180 actcccacca gcgccagaac acagccccccgagctccccg ctaacccagc ccccagaggg 240 gcccaagttc cctcgtgtga agaactgggaggtggggagc atcacctatg acaccctcag 300 cgcccaggcg cagcaggatg ggccctgcaccccaagacgc tgcctgggct ccctggtatt 360 tccacggaaa ctacagggcc ggccctcccccggccccccg gcccctgagc agctgctgag 420 tcaggcccgg gacttcatca accagtactacagctccatt aagaggagcg gctcccaggc 480 ccacgaacag cggcttcaag aggtggaagccgaggtggca gccacaggca cctaccagct 540 tagggagagc gagctggtgt tcggggctaagcaggcctgg cgcaacgctc cccgctgcgt 600 gggccggatc cagtggggga agctgcaggtgttcgatgcc cgggactgca ggtctgcaca 660 ggaaatgttc acctacatct gcaaccacatcaagtatgcc accaaccggg gcaaccttcg 720 ctcggccatc acagtgttcc cgcagcgctgccctggccga ggagacttcc gaatctggaa 780 cagccagctg gtgcgctacg cgggctaccggcagcaggac ggctctgtgc ggggggaccc 840 agccaacgtg gagatcaccg agctctgcattcagcacggc tggaccccag gaaacggtcg 900 cttcgacgtg ctgcccctgc tgctgcaggccccagatgag cccccagaac tcttccttct 960 gccccccgag ctggtccttg aggtgcccctggagcacccc acgctggagt ggtttgcagc 1020 cctgggcctg cgctggtacg ccctcccggcagtgtccaac atgctgctgg aaattggggg 1080 cctggagttc cccgcagccc ccttcagtggctggtacatg agcactgaga tcggcacgag 1140 gaacctgtgt gaccctcacc gctacaacatcctggaggat gtggctgtct gcatggacct 1200 ggatacccgg accacctcgt ccctgtggaaagacaaggca gcagtggaaa tcaacgtggc 1260 cgtgctgcac agttaccagc tagccaaagtcaccatcgtg gaccaccacg ccgccacggc 1320 ctctttcatg aagcacctgg agaatgagcagaaggccagg gggggctgcc ctgcagactg 1380 ggcctggatc gtgcccccca tctcgggcagcctcactcct gttttccatc aggagatggt 1440 caactatttc ctgtccccgg ccttccgctaccagccagac ccctggaagg ggagtgccgc 1500 caagggcacc ggcatcacca ggaagaagacctttaaagaa gtggccaacg ccgtgaagat 1560 ctccgcctcg ctcatgggca cggtgatggcgaagcgagtg aaggcgacaa tcctgtatgg 1620 ctccgagacc ggccgggccc agagctacgcacagcagctg gggagactct tccggaaggc 1680 ttttgatccc cgggtcctgt gtatggatgagtatgacgtg gtgtccctcg aacacgagac 1740 gctggtgctg gtggtaacca gcacatttgggaatggggat cccccggaga atggagagag 1800 ctttgcagct gccctgatgg agatgtccggcccctacaac agctcccctc ggccggaaca 1860 gcacaagagt tataagatcc gcttcaacagcatctcctgc tcagacccac tggtgtcctc 1920 ttggcggcgg aagaggaagg agtccagtaacacagacagt gcaggggccc tgggcaccct 1980 caggttctgt gtgttcgggc tcggctcccgggcatacccc cacttctgcg cctttgctcg 2040 tgccgtggac acacggctgg aggaactgggcggggagcgg ctgctgcagc tgggccaggg 2100 cgacgagctg tgcggccagg aggaggccttccgaggctgg gcccaggctg ccttccaggc 2160 cgcctgtgag accttctgtg tgggagaggatgccaaggcc gccgcccgag acatcttcag 2220 ccccaaacgg agctggaagc gccagaggtaccggctgagc gcccaggccg agggcctgca 2280 gttgctgcca ggtctgatcc acgtgcacaggcggaagatg ttccaggcta caatccgctc 2340 agtggaaaac ctgcaaagca gcaagtccacgagggccacc atcctggtgc gcctggacac 2400 cggaggccag gaggggctgc agtaccagccgggggaccac ataggtgtct gcccgcccaa 2460 ccggcccggc cttgtggagg cgctgctgagccgcgtggag gacccgccgg cgcccactga 2520 gcccgtggca gtagagcagc tggagaagggcagccctggt ggccctcccc ccggctgggt 2580 gcgggacccc cggctgcccc cgtgcacgctgcgccaggct ctcaccttct tcctggacat 2640 cacctcccca cccagccctc agctcttgcggctgctcagc accttggcag aagagcccag 2700 ggaacagcag gagctggagg ccctcagccaggatccccga cgctacgagg agtggaagtg 2760 gttccgctgc cccacgctgc tggaggtgctggagcagttc ccgtcggtgg cgctgcctgc 2820 cccactgctc ctcacccagc tgcctctgctccagccccgg tactactcag tcagctcggc 2880 acccagcacc cacccaggag agatccacctcactgtagct gtgctggcat acaggactca 2940 ggatgggctg ggccccctgc actatggagtctgctccacg tggctaagcc agctcaagcc 3000 cggagaccct gtgccctgct tcatccggggggctccctcc ttccggctgc cacccgatcc 3060 cagcttgccc tgcatcctgg tgggtccaggcactggcatt gcccccttcc ggggattctg 3120 gcaggagcgg ctgcatgaca ttgagagcaaagggctgcag cccactccca tgactttggt 3180 gttcggctgc cgatgctccc aacttgaccatctctaccgc gacgaggtgc agaacgccca 3240 gcagcgcggg gtgtttggcc gagtcctcaccgccttctcc cgggaacctg acaaccccaa 3300 gacctacgtg caggacatcc tgaggacggagctggctgcg gaggtgcacc gcgtgctgtg 3360 cctcgagcgg ggccacatgt ttgtctgcggcgatgttacc atggcaacca acgtcctgca 3420 gaccgtgcag cgcatcctgg cgacggagggcgacatggag ctggacgagg ccggcgacgt 3480 catcggcgtg ctgcgggatc agcaacgctaccacgaagac attttcgggc tcacgctgcg 3540 cacccaggag gtgacaagcc gcatacgcacccagagcttt tccttgcagg agcgtcagtt 3600 gcggggcgca gtgccctggg cgttcgaccctcccggctca gacaccaaca gcccctgaga 3660 gccgcctggc tttcccttcc agttccgggagagcggctgc ccgactcagg tccgcccgac 3720 caggatcagc cccgctcctc ccctcttgaggtggtgcctt ctcacatctg tccagaggct 3780 gcaaggattc agcattattc ctccaggaaggagcaaaacg cctcttttcc ctctctaggc 3840 ctgttgcctc gggcctgggt ccgccttaatctggaaggcc cctcccagca gcggtacccc 3900 agggcctact gccacccgct tcctgtttcttagtccgaat gttagattcc tcttgcctct 3960 ctcaggagta tcttacctgt aaagtctaatctctaaatca agtatttatt attgaagatt 4020 taccataagg gactgtgcca gatgttaggagaactactaa agtgcctacc ccagctc 4077

1-43 (Canceled)
 44. An isolated nucleic acid molecule encoding a nitricoxide synthetase (NOS) polypeptide selected from the group consisting ofa constitutively active NOS polypeptide having an increased activity ofNO production, a NOS polypeptide having an increased rate of NOproduction and a NOS polypeptide having an increased reductase activitywhen compared with the wild-type NOS polypeptide, wherein amino acid S/Tof the consensus sequence motif RXRXXS/T is mutated.
 45. An isolatednucleic acid molecule comprising the sequence as set forth in SEQ ID NO:8 or a variant thereof, wherein said nucleic acid molecule encodes anitric oxide synthetase (NOS) polypeptide selected from the groupconsisting of a constitutively active NOS polypeptide having anincreased activity of NO production, a NOS polypeptide having anincreased rate of NO production and a NOS polypeptide having anincreased reductase activity when compared with the wild-type NOSpolypeptide, wherein amino acid S/T of the consensus sequence motifRXRXXS/T is mutated.
 46. The isolated nucleic acid molecule of claim 45,wherein the NOS polypeptide comprises a substituted amino acid residuecorresponding to residue 1179 of bovine eNOS, residue 1177 of humaneNOS, residue 1412 of rat nNOS, or residue 1415 of human nNOS.
 47. Theisolated nucleic acid molecule of claim 46, wherein the substitutedamino acid residue corresponding to residue 1179 of bovine eNOS, residue1177 of human eNOS, residue 1412 of rat nNOS, or residue 1415 of humannNOS, mimics a phosphoserine.
 48. The isolated nucleic acid molecule ofclaim 46, wherein a serine corresponding to residue 1179 of bovine eNOS,residue 1177 of human eNOS, residue 1412 of rat nNOS, or residue 1415 ofhuman nNOS, has been substituted with an aspartic acid residue or aglutamic acid residue.
 49. The isolated nucleic acid molecule of claim46, wherein the substituted amino acid comprises an R group that mimicsa phosphate group.
 50. The isolated nucleic acid molecule of claim 46,wherein the substituted amino acid comprises a negatively charged Rgroup.
 51. An isolated nucleic acid molecule encoding a human eNOSpolypeptide as set forth in SEQ ID NO. 3, wherein the eNOS polypeptideis selected from the group consisting of a constitutively active NOSpolypeptide having an increased activity of NO production, a NOSpolypeptide having an increased rate of NO production and a NOSpolypeptide having an increased reductase activity when compared withthe wild-type NOS polypeptide, wherein amino acid S/T of the consensussequence motif RXRXXS/T is mutated.
 52. An isolated nucleic acidmolecule encoding a human eNOS polypeptide which comprises a substitutedamino acid residue corresponding to residue 1177 of human eNOS as setforth in SEQ ID NO.
 5. 53. The isolated nucleic acid molecule of claim52, wherein the substituted amino acid is an aspartic acid residue asset forth in SEQ ID NO. 6 or a glutamic acid residue as set forth in SEQID NO. 7.